EP3768284B1 - Compositions of entrecoccus flagellin for use in therapy - Google Patents

Compositions of entrecoccus flagellin for use in therapy Download PDF

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EP3768284B1
EP3768284B1 EP19714132.8A EP19714132A EP3768284B1 EP 3768284 B1 EP3768284 B1 EP 3768284B1 EP 19714132 A EP19714132 A EP 19714132A EP 3768284 B1 EP3768284 B1 EP 3768284B1
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Prior art keywords
flagellin
compositions
polypeptide
flagellin polypeptide
seq
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English (en)
French (fr)
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EP3768284A1 (en
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Imke Elisabeth MULDER
Emma RAFTIS
Emma Elizabeth Clare HENNESSY
Delphine Louise Claudette LAUTE-CALY
Philip COWIE
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4D Pharma Research Ltd
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4D Pharma Research Ltd
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Priority claimed from GBGB1804384.4A external-priority patent/GB201804384D0/en
Priority claimed from GBGB1809953.1A external-priority patent/GB201809953D0/en
Priority claimed from GBGB1811900.8A external-priority patent/GB201811900D0/en
Priority claimed from GBGB1812378.6A external-priority patent/GB201812378D0/en
Priority claimed from GBGB1813444.5A external-priority patent/GB201813444D0/en
Priority claimed from GBGB1813423.9A external-priority patent/GB201813423D0/en
Priority claimed from GBGB1816834.4A external-priority patent/GB201816834D0/en
Priority claimed from GBGB1817641.2A external-priority patent/GB201817641D0/en
Priority claimed from GBGB1901199.8A external-priority patent/GB201901199D0/en
Priority claimed from GBGB1901218.6A external-priority patent/GB201901218D0/en
Priority claimed from GBGB1901993.4A external-priority patent/GB201901993D0/en
Priority claimed from GBGB1901992.6A external-priority patent/GB201901992D0/en
Priority to SI201930190T priority Critical patent/SI3768284T1/sl
Priority to RS20220288A priority patent/RS63090B1/sr
Application filed by 4D Pharma Research Ltd filed Critical 4D Pharma Research Ltd
Priority to EP22150203.2A priority patent/EP4046644A1/en
Priority to HRP20220368TT priority patent/HRP20220368T1/hr
Priority to PL19714132T priority patent/PL3768284T3/pl
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention is in the field of compositions comprising flagellin polypeptides from bacterial strains and the use of such compositions in the treatment of disease.
  • the human intestine is thought to be sterile in utero, but it is exposed to a large variety of maternal and environmental microbes immediately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host genotype, all of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-like [1].
  • the human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two major bacterial divisions, the Bacteroidetes and the Firmicutes [2].
  • the successful symbiotic relationships arising from bacterial colonization of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions.
  • the enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host.
  • the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animals which have an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [3-5].
  • Flagellin is one of the principal components of the bacterial flagellum.
  • the flagellum is a lash-like appendage that provides the bacterium with motility and can act as a sensory organelle.
  • the structure and composition of bacterial flagellin has been comprehensively studied in model organisms, such as Salmonella typhimurium and E. coli.
  • Flagellin of Salmonella typhimurium and E. coli activate the immune system via the TLR5 receptor, which leads the activation of a variety of proinflammatory and immune response genes [15]. It has been suggested that TLR5 agonists, such as Salmonella typhimurium flagellin can act as anti-cancer agents [16].
  • Salmonella typhimurium engineered to express Vibrio vulnifificus flagellin has also been proposed to act as an anti-cancer agent [17]. Additionally, it has been suggested that flagellin peptides from Salmonella typhimurium, Vibrio cholera and Shigella dysenteriae can be used to produce vaccines against infectious disease such as typhoid, cholera and dysentery when expressed by intestinal bacteria which have been genetically modified [18].However, flagellin from other species of bacteria remain poorly characterised and such other flagellin may have different effects or uses.
  • Streptococcus and Veillonella strains and to a lesser extent, Enterococcus and Lactobaccillus strains have been suggested to have immunomodulatory effects, with varying effects on different cytokines in vitro.
  • JP2017/506212 discloses algal based edible vaccines.
  • WO2009/130618 discloses flagellin polypeptide vaccines.
  • WO2017/085520 discloses compositions comprising bacterial strains. Lorena Roman et al. discuss the effect of probiotic Enterococcus gallinarum L-1 on the innate immune parameters of outstanding species to marine aquaculture. There is a requirement in the art for new methods of treating diseases. There is also a requirement for the potential effects of gut bacteria to be characterised so that new therapies can be developed.
  • flagellin polypeptides from the genus Enterococcus are useful in therapy.
  • flagellin polypeptides from the genus Enterococcus can strongly activate TLR5 responses at levels significantly higher than the well-known TLR5 agonist Salmonella. typhimurium flagellin.
  • the invention provides flagellin polypeptides from the species Enterococcus gallinarum for use in therapy.
  • the flagellin polypeptide is from the strain deposited at NCIMB under accession number NCIMB 42488.
  • the flagellin polypeptide is SEQ ID NO:1. The examples show that flagellin polypeptides from the strain MRx0518 (the strain deposited under accession number NCIMB 42488) can more strongly activate a TLR5 response compared to flagellin polypeptides from other strains of Enterococcus gallinarum. Thus, flagellin polypeptides from the strain NCIMB 42488 are particularly effective for use in therapy.
  • the flagellin polypeptide is in a monomeric form, which allows the polypeptide to more effectively bind with a TLR5 receptor.
  • flagellin polypeptides from the strain NCIMB 42488 are particularly effective at activating a TLR5 response.
  • Flagellin polypeptides from the strain NCIMB 42488 may not contain a D3 domain and the inventors have noted that the majority of the sequence variation between different flagellin polypeptides is observed in the D2-D3 region.
  • the invention provides flagellin polypeptides from the genus Enterococcus or species Enterococcus gallinarum that do not contain a D3 domain.
  • the flagellin polypeptide is part of a bacterial flagellar assembly.
  • the invention provides a bacterial flagellar assembly comprising a flagellin polypeptide as described herein.
  • the bacterial flagellar assembly contains the proteins FliL and FlaG. FliL and FlaG proteins are present in the bacterial flagellar assembly of MRx0518. The examples show that flagellin polypeptide from MRx0518 are particularly effective at activating a TLR5 response.
  • the invention provides a fragment of a flagellin polypeptide from the genus Enterococcus, for use in therapy, wherein the fragment comprises amino acids 87-96, 165-272 and 290-295 of SEQ ID NO: 1.
  • the invention provides a fusion polypeptide comprising a flagellin polypeptide as described herein for use in therapy.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a fragment of a flagellin polypeptide from the genus Enterococcus, wherein the fragment a) comprises amino acids 87-96, 165-272 and 290-295 of SEQ ID NO: 1 and one or more pharmaceutically acceptable excipients or carriers; or b) comprises amino acids 2-32, 87-96, 165-272, 234-358, 290-295 from of SEQ ID NO:1 and one or more pharmaceutically acceptable excipients or carriers.
  • the invention also provides a polynucleotide sequence that encodes a flagellin polypeptide from the genus Enterococcus for use in therapy.
  • the polynucleotide sequence encodes a flagellin polypeptide from the species Enterococcus gallinarum for use in therapy.
  • the polynucleotide sequence encodes a flagellin polypeptide from the strain MRx0518 for use in therapy.
  • the invention provides a host cell expressing a recombinant flagellin polypeptide from the genus Enterococcus, for use in therapy.
  • the invention also provides a host cell comprising a recombinant polynucleotide sequence that encodes a flagellin polypeptide from the genus Enterococcus, for use in therapy.
  • the invention provides a vector or a plasmid comprising a polynucleotide sequence according to the invention.
  • compositions for use in therapy wherein the composition comprises flagellin polypeptides as described herein.
  • the invention also provides compositions comprising a polynucleotide sequence encoding a flagellin polypeptide as described herein for use in therapy.
  • the invention also provides a host cell that expresses a flagellin polypeptide as described herein for use in therapy.
  • the invention also provides compositions for use in therapy wherein the composition comprises a host cell, wherein the host cell comprises a polynucleotide according to the invention.
  • compositions comprising Enterococcus gallinarum strains are effective at treating and preventing cancer [50].
  • flagellin polypeptides from the species Enterococcus gallinarum are able to stimulate a strong TLR5 response, which may contribute to the anti-cancer activity of Enterococcus strains.
  • flagellin polypeptides from the genus Enterococcus, and in particular the species Enterococcus gallinarum are useful in therapy, and particularly effective in treating and preventing cancer.
  • compositions comprising flagellin polypeptides from the genus Enterococcus are for use in treating immunogenic tumours and / or solid tumours.
  • compositions comprising flagellin polypeptides from the genus Enterococcus are for use in a method of treating or preventing lung cancer, breast cancer, liver cancer or colon cancer.
  • Compositions comprising flagellin polypeptides from the genus Enterococcus may be particularly effective for reducing tumour size or preventing tumour growth in the treatment of cancer.
  • composition for use in the invention is administered by injection, preferably subcutaneously or alternatively intravenously, or intraperitoneally.
  • the composition for use in the invention is for oral administration.
  • the composition for use in the invention comprises one or more pharmaceutically acceptable excipients or carriers.
  • compositions comprising a flagellin polypeptide from the genus Enterococcus, preferably from the species Enterococcus gallinarum that can be used in stimulating the immune system and treating and preventing disease.
  • the inventors have identified that such compositions can potently activate the immune system and can treat cancer, which indicates that they may able to also treat other diseases where activation of the immune system may be useful.
  • the invention therefore provides a composition comprising a flagellin polypeptide from the genus Enterococcus, for use in stimulating the immune system in subject.
  • the invention provides a composition comprising a flagellin polypeptide from the genus Enterococcus, for use in treating, preventing or delaying immunosenescence.
  • the invention provides a composition comprising a flagellin polypeptide from the genus Enterococcus, for use as a vaccine adjuvant.
  • the invention provides a composition comprising a flagellin polypeptide from the genus Enterococcus, for use in enhancing a cell therapy, such as CAR-T.
  • Flagellin a principal component of bacterial flagella, stimulates host defence in a variety of organisms, including mammals, insects, and plants.
  • the structure and composition of bacterial flagellin has been comprehensively studied in model organisms such as S. typhimurium and E. coli.
  • Flagellin can exist either as a monomeric protein or can be polymerised to form a flagellum filament.
  • Bacterial flagellum filaments in Salmonella are composed of approximately 20,000 flagellin protein units. Flagellum filaments form part of a fully assembled bacterial flagellum.
  • Flagellin proteins generally contain 3-4 domains (D0, D1, D2 and D3), with the D0 and D1 domains consisting of a fold over from the N- and C-termini [31]. During flagellum filament assembly, the D0 and D1 domains are buried within the flagellum filament with the D2 and/or D3 domains facing outwards ( Figure 1 ).
  • TLR5 recognition site is generally not accessible in the flagellar filament.
  • Flagellin interacts more effectively with TLR5 in its monomeric form, as the TLR5 binding sites are not accessible when assembled in a filament [30].
  • the central region of the flagellin protein displays sequence variability and has not been studied extensively but is considered to be an antigenic and immunogenic region of the protein.
  • Figure 2 is a sequence alignment of flagellin proteins from the genus Enterococcus with the extensively studied flagella protein from S. typhimurium .
  • the majority of the sequence variation between the flagellin protein from S. typhimurium and the flagellin proteins from the genus Enterococcus lies within the central region (D2-D3 domain).
  • the majority of the sequence variation between different Enterococcus gallinarum strains is also observed in this region, for example between flagellin proteins from the strains MRx0518 and DSM100110 ( Figure 3 ).
  • the flagellum filament is a component of the bacterial flagellum.
  • the genes known to be involved in bacterial flagellar assembly are summarised in Figure 4 . Those shaded in grey are present in Enterococcus gallinarum. .
  • the flagellin proteins are encoded by FliC, which is also interchangeably referred to herein as FlaA.
  • the invention provides a flagellin polypeptide from the genus Enterococcus for use in therapy.
  • the invention provides a flagellin polypeptide from the species Enterococcus gallinarum for use in therapy.
  • the invention provides a flagellin polypeptide from the strain MRx0518 for use in therapy.
  • the examples demonstrate that flagellin polypeptides from the genus Enterococcus can activate a strong TLR5 response, which shows flagellin polypeptides may be useful in therapy, and in particular useful in treating cancer.
  • the invention provides a flagellin polypeptide from the genus Enterococcus for use in a method of treating or preventing cancer.
  • the invention provides a flagellin polypeptide from the species Enterococcus gallinarum for use in a method of treating or preventing cancer.
  • the invention provides a flagellin polypeptide from the strain MRx0518 for use in a method of treating or preventing cancer.
  • the examples demonstrate that flagellin polypeptides from the genus Enterococcus are able to induce strong TLR5 responses, which is useful in treating and preventing cancer.
  • the examples show that flagellin polypeptides from the species Enterococcus gallinarum and especially strain MRx0518 produce a very high TLR5 response.
  • the invention provides a flagellin polypeptide from the species Enterococcus gallinarum or the species Enterococcus casseliflavus, for use in therapy, in particular for treating or preventing cancer.
  • the flagellin polypeptide is from the species Enterococcus casseliflavus.
  • the flagellin polypeptide is from the species Enterococcus gallinarum.
  • MRx0518 is a strain of Enterococcus gallinarum bacterium deposited under accession number NCIMB 42488. Strain MRx0518 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 16th November 2015 as "Enterococcus sp" and was assigned accession number NCIMB 42488.
  • the invention provides a flagellin polypeptide with at least 75% sequence identity to the flagellin polypeptide from the strain MRx0518 for use in therapy, and in particular in treating or preventing cancer.
  • the examples show that such flagellin polypeptides are especially effective.
  • the flagellin polypeptide having a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:1.
  • the flagellin polypeptide for use in the invention comprises or consists of SEQ ID NO:1.
  • the flagellin polypeptide has a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:2.
  • the flagellin polypeptide for use in the invention may comprise or consist of SEQ ID NO:2. The examples also demonstrate that such polypeptides are useful.
  • the flagellin polypeptide has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to one of SEQ ID NO:3-42. In preferred embodiments, the flagellin polypeptide has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to one of SEQ ID NO:3-16. In preferred embodiments, the flagellin polypeptide is from the species Enterococcus gallinarum.
  • the flagellin for use in the invention binds TLR5, for example with a K D value of at least 10 -2 , at least 10 -3 , at least 10 -4 , at least 10 -5 , at least 10 -6 , at least 10 -7 , at least 10 -8 , at least 10 -9 , at least 10 -10 , or at least 10 -11 .
  • Binding to TLR5 may be measured by any appropriate method known in the art, including electrochemical impedance spectroscopy (EIS), scanning electrochemical microscopy (SECM) [36], or native PAGE and gel-filtration chromatography analyses using a recombinant TLR5 protein [37].
  • the flagellin polypeptides for use in the invention can contain four domains, D0, D1, D2 and D3. In preferred embodiments, the flagellin polypeptide does not comprise a D3 domain. In certain embodiments the flagellin polypeptide for use in the invention consists of three domains, D0, D1, D2. The location of the D0, D1 and D2 domains in E. gallinarum flagellin polypeptide are shown in Figure 3 . The predicted shape of the flagellin polypeptide from the strain MRx0518 (FliC MRx0518 ) is shown in Figure 5 .
  • the predicted structure of FliC MRx0518 was generated using the Phyre2 tool which predicts protein structure based on homology with publicly available structures [38] and the graphic was generated with the UCSF Chimera package [39].
  • the predicted lack of D3 domain leads to a conformation change in the protein structure compared to flagellin polypeptide from S. typhimurium SL1344 (GenBank Accession No. CBW17983) .
  • the predicted lack of a D3 domain may lead to the TLR5 recognition site being more exposed, which leads to an increase in the ability of the flagellin polypeptide to activate a TLR5 response.
  • the predicted lack of a D3 domain may also affect the ability of flagellin polypeptide polymerise into a flagellum filament. Flagellin polypeptide activates TLR5 responses more effectively in the monomeric form, therefore preventing or reducing the polymerisation of flagellin polypeptides is beneficial.
  • the flagellin polypeptides for use in the invention may contain a D1 domain, which may be required for TLR5 recognition and interaction.
  • the flagellin polypeptide with a D1 domain has at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to residues 43-164 and 273-317 in SEQ ID NO:1.
  • the flagellin polypeptide has a TLR5 recognition site in the D1 domain.
  • the TLR recognition site in flagellin polypeptides from the species Enterococcus gallinarum is located at position 87-96 and 290-295 in SEQ ID NO:1.
  • the TLR5 recognition site of the flagellin polypeptide has at least 99%, 99.5% or 99.9% identity to residues 87-96 and 290-295 in SEQ ID NO:1.
  • the flagellin polypeptide comprises the TLR5 recognition site of residues 87-96 and 290-295 in SEQ ID NO:1.
  • the flagellin polypeptide for use in the invention may also contains a D0 domain, which can affect TLR5 signalling.
  • the flagellin polypeptide with at D0 domain has at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to residues 2-32 and 234-358 in SEQ ID NO:1.
  • the flagellin polypeptide for use in the invention may also contain a D2 domain.
  • the majority of the sequence variation between the flagella polypeptides from S. typhimurium and the genus Enterococcus lie within the central region (D2-D3 domain).
  • the examples show that flagellin polypeptides from the species Enterococcus gallinarum are able to produce stronger TLR5 response at a lower dosage compared to flagellin polypeptide from S. typhimurium.
  • the sequence variation in the D2 or D3 domain may contribute to the increased ability of flagellin polypeptides from the species Enterococcus gallinarum to activate a TLR5 response.
  • the flagellin polypeptide also contains a D0 domain, which can affect TLR5 signalling.
  • the flagellin polypeptide with at D2 domain has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity to residues 165-272 in SEQ ID NO:1.
  • Flagellin polypeptide can exist as a monomer or in a polymerised form as a flagellum filament. This filament is part of the bacterial flagellum. In certain embodiments, the flagellin polypeptide is arranged in flagellum filament. The invention also relates to flagellin polypeptides for use in therapy not arranged in flagellum filament. In preferred embodiments, the flagellin polypeptide is in its monomeric form. The monomeric form of the flagellin polypeptide is advantageous as it can interact more strongly with TLR5. In embodiments, the flagellin is not glycosylated, to aid its shedding and disaggregation into the monomeric form.
  • the flagellin polypeptide is part of a bacterial flagellum. In certain embodiments, the flagellin polypeptide is part of a bacterial flagellum that contains one or more proteins selected from FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS.
  • the bacterial flagellar assembly contains FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS.
  • the bacterial flagellar assembly contains the proteins FliL and FlaG.
  • FliL is flagellar basal body-associated protein, which controls the rotational direction of the flagella during chemotaxis and FlaG regulates flagellin assembly along with FlaF.
  • FliL and FlaG proteins are present in MRx0518. The examples show that flagellin polypeptide from MRx0518 can activate a stronger TLR5 response compared to flagellin polypeptide from another Enterococcus gallinarum species.
  • the flagellin polypeptides may be recombinantly expressed, or they may be isolated from Enterococcus cells. In certain embodiments, the flagellin polypeptide is not attached to an Enterococcus cell. In certain embodiments, the flagellin polypeptide is in a composition that is substantially free of bacterial cells.
  • the flagellin polypeptides have been heat treated, and optionally denatured. The examples demonstrate that such flagellin polypeptides are still potently effective.
  • the flagellin polypeptides are heat-stable to 80°C, for example can maintain activity following heating to 80°C for, for example, 40 mins.
  • the flagellin polypeptides are digested by trypsin. In other words, in certain embodiments, the flagellin polypeptides contain trypsin cleavage sites.
  • the MRX518 flagellin is predicted to contain 36 trypsin cleavage sites.
  • a flagellin polypeptide for use according to the invention does not have to function as a normal flagellin polypeptide in order to be considered a flagellin polypeptide.
  • a flagellin polypeptide for use according to the invention may contain mutations or deletions that ablate certain activities.
  • a flagellin polypeptide for use in the invention is a TLR5 agonist.
  • the flagellin polypeptides are not able to polymerise into a flagellum filament.
  • the flagellin polypeptides are not able to form part of the bacterial flagellar assembly.
  • polypeptides have sequence identity to SEQ ID NO:1, as set out above, or comprise or consist of SEQ ID NO:1.
  • the invention also provides compositions comprising a flagellin polypeptide from the genus Enterococcus for use in therapy.
  • the composition comprises a flagellin polypeptide from the species Enterococcus gallinarum for use in therapy.
  • the composition comprises a flagellin polypeptide from the strain MRx0518 for use in therapy.
  • the flagellin polypeptide is of the species Enterococcus caselliflavus.
  • Enterococcus caselliflavus is highly similar to Enterococcus gallinarum and is also flagellated.
  • Flagellin polypeptides with sequence identity to those tested in the examples are expected to also be useful in therapy and are encompassed by the invention, as set out in the preceding section.
  • the invention also provides fragments of a flagellin polypeptide from the genus Enterococcus, for use in therapy, wherein the fragment comprises amino acids 87-96, 165-272 and 290-295 of SEQ ID NO:1.
  • any fragment is at least 200, 250, 300 or 350 amino acids in length.
  • Preferred fragments comprise one or more of the following stretches of amino acids of SEQ ID NO:1: 2-32, 87-96, 165-272, 234-358, 290-295.
  • a fragment for use according to the invention will usually (i) have at least w% sequence identity to SEQ ID NO: 1 and/or (ii) comprise of a fragment of at least x contiguous amino acids from SEQ ID NO: 1.
  • the value of w is at least 85 (e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more).
  • the value of x is at least 7 (e.g.
  • the fragment will usually include an epitope from SEQ ID NO: 1.
  • the fragment will usually be able to bind to TLR5.
  • fragments for use according to the invention will usually (i) have at least w% sequence identity to SEQ ID NO: 2 and/or (ii) comprise of a fragment of at least x contiguous amino acids from SEQ ID NO: 2.
  • the value of w is at least 85 (e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more).
  • the value of x is at least 7 (e.g.
  • the fragment will usually include an epitope from SEQ ID NO: 2.
  • the fragment will usually be able to bind to TLR5.
  • fragments for use according to the invention will usually (i) have at least w% sequence identity to one of SEQ ID NO: 3-42 and/or (ii) comprise of a fragment of at least x contiguous amino acids from one of SEQ ID NO: 3-42.
  • the value of w is at least 85 (e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more).
  • the value of x is at least 7 (e.g.
  • the fragment will usually include an epitope from one of SEQ ID NO: 3-42.
  • the fragment will usually be able to bind to TLR5.
  • Fusions comprising the flagellin polypeptides as described herein are also expected to be useful in therapy. Therefore, the invention provides a fusion polypeptide comprising a flagellin polypeptide as described above for use in therapy, and in particular for use in treating cancer.
  • Preferred fusion partners include targeting moieties and moieties intended to prolong half-life.
  • the fusion polypeptide comprises a flagellin polypeptide as described above and a polypeptide selected from the group consisting of an Fc region, transferrin, albumin, recombinant PEG, a homoamino acid polymer, a proline-alanine-serine polymer, an elastin-like peptide, or carboxy-terminal peptide [CTP; of chorionic gonadotropin(CG) b-chain].
  • a polypeptide selected from the group consisting of an Fc region, transferrin, albumin, recombinant PEG, a homoamino acid polymer, a proline-alanine-serine polymer, an elastin-like peptide, or carboxy-terminal peptide [CTP; of chorionic gonadotropin(CG) b-chain].
  • flagellin polypeptides for use in the invention may also be fused or conjugated to non-polypeptide moieties in order to improve their characteristics and therapeutic utility.
  • the flagellin polypeptides as described above are covalently linked to PEG or hyaluronic acid.
  • the flagellin polypeptides for use in the invention may be fused or conjugated to an antigen, for example, for use as a vaccine. Presenting an antigen in close proximity to the flagellin may maximise their immunostimulatory activities and further enhance the protective immune response generated against the antigen.
  • manufacturing and delivering therapeutics comprising an antigen and a flagellin as described herein may be more efficient and effective when they are fused or conjugated. Therefore, in certain embodiments, the fusion polypeptide comprising a flagellin polypeptide as described above and an antigen.
  • the invention provides a flagellin polypeptide as described above conjugated to an antigen.
  • Preferred antigens for including in the fusions and conjugates for use in the invention are pathogen antigens and tumour antigens.
  • An antigen will elicit an immune response specific for the antigen that will be effective for protecting against infection with the pathogen or attacking the tumour.
  • Antigens may be, for example, peptides or polysaccharides.
  • Exemplary antigens for use with the fusions and conjugates for use in the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens.
  • antigens for use with the fusions and conjugates for use in the invention include glycoprotein and lipoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit forming substances, for example nicotine, alcohol or opiates.
  • MAGE Carcinoembryonic antigen
  • MUC-1 MUC-1
  • HER2 sialyl-Tn
  • STn sialyl-Tn
  • hTERT human telomerase reverse transcriptase
  • WT1 Wilms tumour gene
  • CA-125 CA-125
  • PSA prostate-
  • Fusion polypeptides for use in the invention may include a linker sequence between the flagellin and the antigen.
  • Preferred linker sequences include peptides that comprise (Gly4)n motif(s), a (Gly4Ser)n motif(s), and Ser(Gly4Ser)n motif(s).
  • Linker sequences may be 1-50, 1-30, 1-20, 5-20, 5-10 or 10-20 amino acids in length. Linker sequences will allow the flagellin and the fusion partner, for example the antigen, to perform their roles and, for example, be accessed by the immune system.
  • the invention provides a fusion polypeptide comprising a flagellin polypeptide as described above, a linker sequence, and an antigen, such as an antigen derived from a pathogen, or a tumour antigen for use in therapy.
  • Flagellin polypeptides for use in the invention may be particularly useful when conjugated to non-polypeptide moieties such as polysaccharide antigens. Suitable conjugation methods include using carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, CDAP or TSTU.
  • the invention also provides a polynucleotide sequence that encodes a flagellin polypeptide from the genus Enterococcus for use in therapy.
  • the polynucleotide sequence encodes a flagellin polypeptide from the species Enterococcus gallinarum for use in therapy.
  • the polynucleotide sequence encodes a flagellin polypeptide from the strain MRx0518 for use in therapy.
  • the examples demonstrate that flagellin polypeptides from the genus Enterococcus can activate a strong TLR5 response, so polynucleotides encoding such flagellin polypeptides may be useful in therapy, and in particular useful in treating cancer.
  • the invention also provides a polynucleotide sequence that encodes a flagellin polypeptide from the genus Enterococcus for use in a method of treating or preventing cancer.
  • the invention provides a polynucleotide sequence that encodes a flagellin polypeptide from the species Enterococcus gallinarum for use in a method of treating or preventing cancer.
  • the invention provides a polynucleotide sequence that encodes a flagellin polypeptide from the strain MRx0518 for use in a method of treating or preventing cancer.
  • the examples demonstrate that flagellin polypeptide from the genus Enterococcus are able to induce strong TLR5 responses, which is useful in treating and preventing cancer.
  • the examples show that flagellin polypeptides from the species Enterococcus gallinarum and especially strain MRx0518 produce a very high TLR5 response.
  • the polynucleotide of the invention for use in therapy encodes SEQ ID NO:1, or a polypeptide sequence with 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:1.
  • the polynucleotide can encode a flagellin with the particular D0, D1 and/or D2 sequences set out above, which are shown in the examples to be useful.
  • the polynucleotide of the invention for use in therapy encodes SEQ ID NO:2, or a polypeptide sequence with 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:2.
  • the polynucleotide can encode a flagellin with the particular D0, D1 and/or D2 sequences set out above, which are shown in the examples to be useful.
  • the polynucleotide of the invention for use in therapy encodes one of SEQ ID NO: 3-42, or a polypeptide sequence with 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to one of SEQ ID NO: 3-42.
  • the polynucleotide can encode a flagellin with the particular D0, D1 and/or D2 sequences set out above, which are shown in the examples to be useful.
  • Nucleic acids for use in the invention can take various forms (e.g. single stranded, double stranded, vectors etc.). Nucleic acids for use in the invention may be circular or branched, but will generally be linear.
  • nucleic acid includes both single-stranded and double-stranded nucleotide polymers.
  • the nucleotides comprising the nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • Nucleic acids may be prepared in many ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc.
  • nucleases e.g. restriction enzymes
  • ligases or polymerases e.g. using ligases or polymerases
  • the nucleic acids for use in the invention comprise a sequence which encodes at least one polypeptide flagellin polypeptide as described herein.
  • the nucleic acids will be in recombinant form, i.e. a form which does not occur in nature.
  • the nucleic acid may comprise one or more heterologous nucleic acid sequences (e.g. a sequence encoding another antigen and/or a control sequence such as a promoter or an internal ribosome entry site) in addition to the sequence encoding at least flagellin polypeptide.
  • the nucleic acid encodes a flagellin polypetide as discussed above and an antigen, such as a pathogen antigen or a tumour antigen.
  • the nucleic acid also encodes a linker sequence.
  • the invention also provides host cells expressing a flagellin polypeptide as described herein, for use in therapy.
  • the examples demonstrate that Enterococcus strains such as MRx0518, which express Enterococcus flagellin, can activate TLR5 and therefore may have potent therapeutic effects.
  • Host cells expressing flagellin polypeptides may be useful for producing flagellin polypeptide for inclusion in a therapeutic composition, or such host cells may be useful for administering to a patient, wherein they express the flagellin polypeptide to exert a direct therapeutic effect.
  • the host cell for use in the invention expresses the flagellin polypeptide as described herein recombinantly.
  • the host cell comprises a heterologous polynucleotide sequence that encodes the flagellin polypeptide as described herein.
  • the host cell is not of the genus Enterococcus, is not of the species Enterococcus gallinarum, or is not of the strain MRx0518.
  • the host cell for expressing recombinant Enterococcus flagellin polypeptides is E. coli.
  • the host cell is for administration to a patient
  • the host cell is a probiotic strain, such as a Lactobacillus or Bifidobacterium strain.
  • the host cell comprises a polynucleotide sequence that encodes a flagellin polypeptide from the genus Enterococcus. In certain embodiments, the host cell comprises a polynucleotide sequence that encodes a flagellin polypeptide from the species Enterococcus gallinarum. In preferred embodiments, the host cell comprises a polynucleotide sequence that encodes a flagellin polypeptide from the strain MRx0518. In certain embodiments, the host cell comprises a vector, wherein the vector comprises a polynucleotide sequence that encodes a flagellin polypeptide from the genus Enterococcus.
  • the host cell comprises a polynucleotide sequence that encodes one or more of, preferably all of, the bacterial flagellar assembly genes FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS.
  • the bacterial flagellar assembly genes FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS.
  • the host cell expresses one or more of, preferably all of,the polypeptides FliD, FlgL, FlgK, FlgE, FliK, FlgD, FlgG, MotB, MotA, FliF, FliG, FliM, FliN, FlhA FlhB, FliH, FliI, FliO, FliP, FliQ, FliR, FliL, FliJ and FliS.
  • the host cell comprises a polynucleotide sequence that encodes a bacterial flagellar assembly comprising FliL and FlaG.
  • the host cell expresses the polypeptides FliL and FlaG.
  • any cell expressing a flagellin polypeptide as described herein, and in particular any cell for administration to a patient is effective at shedding flagellin polypeptide.
  • the examples demonstrate that such cells induce a stronger TLR5 response.
  • Flagellin can be "actively" shed during flagellum assembly, through leakage of its monomeric units. However, in most cases monomeric flagellin arise from the passive degradation of detached flagella. Flagellar detachment can occur following mechanical strain or as a voluntary process. In some bacterial species flagellin is glycosylated to enhance filament stability and prevent uncontrolled shedding, for example in Shewanella oneidensis [40]. In preferred embodiments, the flagellin expressed by the host cell is not glycosylated, to aid its shedding and disaggregation into the monomeric form.
  • the flagellin polypeptide and / or the bacterial flagellar assembly as described herein is easily shed from a host cell.
  • the ease of shedding of flagellin polypeptides and / or flagellar assemblies can be determined by separately growing i) cells expressing the flagellin as described herein and ii) one or more cells (e.g. 1, 2, 3, 4, 5, 10 or more than 10) expressing reference flagellins derived from the same or different strains belonging to the same bacterial species as the flagellin as described herein a) in a stationary phase for 18 hours using a 1% inoculum of the respective cells, then b) a late-log phase in which 10% inoculum of cells from the stationary phase are grown for 3 hours.
  • cells e.g. 1, 2, 3, 4, 5, 10 or more than 10
  • 10ml cultures of the cells expressing the flagellin as described herein and of the cells expressing reference flagellins can then be centrifuged at 5000xg for 5 minutes at room temperature to remove the bacterial pellet.
  • the resulting supernatants can then be filtered (0.22um) under sterile conditions.
  • the concentration of the flagellin protein in the supernatant derived from cells expressing the flagellin as described herein will be higher (e.g.
  • the host cell for use in the invention releases flagellin polypeptides and/or flagellar assemblies into its environment with requiring chemical or physical disruption.
  • culturing a host cell for use in the invention under conditions suitable for expression of the flagellin polypeptide results in flagellin polypeptide being released from the cell into its environment.
  • the examples demonstrate that cells expressing flagellin polypeptides as described herein induce a stronger TLR5 response.
  • the host cells for use in the invention are good shedders of the bacterial flagellar assembly.
  • the host cells are good shedders of bacterial flagellum filaments and in particular good shedders of flagellin polypeptides.
  • the bacterial flagellar assembly as described herein actively sheds monomeric flagellin.
  • the flagellum filament as described herein actively sheds monomeric flagellin.
  • Monomeric flagellin is more effective at inducing TLR5 responses.
  • the disclosure provides a method for obtaining a flagellin polypeptide or flagellar assembly as described herein comprising culturing a cell expressing a flagellin polypeptide or flagellar assembly as described herein and culturing said flagellin polypeptide or flagellar assembly, without performing any step to remove separate flagellin polypeptide or flagellar assembly from the cell surface.
  • flagellum (or flagellin) shedding is not currently associated with the expression of a specific subset genes and is likely to be heavily influenced by environmental conditions.
  • a number of genes have been shown to be involved in flagellum rigidity and stability.
  • flagellum rigidity and stability For example in Salmonella, fliF and fliL mutants have been shown to shed flagella more easily when grown in viscous media [41,42]. In both cases, flagella broke near the distal rod [43]. Therefore, in preferred embodiments, the host cell does not express fliF and/or fliL. In other embodiments, the host cell does express fliL.
  • the host cells for use in the invention have been heat treated.
  • the examples demonstrate that cells expressing flagellin polypeptides as described herein are still potently effective after heat treatment.
  • the host cells for use in the invention are heat-stable to 80°C, for example can maintain activity following heating to 80°C for, for example, 40 mins.
  • the disclosure further provides methods of producing the flagellin polypeptides as described herein by culturing the host cells under conditions permitting production of the flagellin polypeptides, and recovering the flagellin polypeptides so produced.
  • the host cells for use in the invention additionally expresses one or more antigens.
  • the antigen will be expressed recombinantly and will be heterologous to the host cell.
  • the antigen may be part of a fusion polypeptide expressed with the flagellin polypeptide as described herein, or the host cell may separately express the flagellin and the antigen from different open reading frames. Therefore, the invention provides a host cell that expresses a flagellin polypeptide as described herein and a heterologous antigen for use in therapy.
  • antigens for use with the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens.
  • Further antigens for expressing in a host cell with a flagellin for use in the invention include glycoprotein and lipoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit forming substances, for example nicotine, alcohol or opiates.
  • MAGE Carcinoembryonic antigen
  • CEA Carcino
  • the nucleic acid for use in the invention may be part of a vector i.e. part of a nucleic acid construct designed for transduction/transfection of one or more cell types.
  • Vectors may be, for example, "expression vectors” which are designed for expression of a nucleotide sequence in a host cell, or "viral vectors” which are designed to result in the production of a recombinant virus or virus-like particle.
  • sequence or chemical structure of the nucleic acid may be modified compared to a naturally-occurring sequence which encodes a flagellin polypeptide.
  • the sequence of the nucleic acid molecule may be modified, e.g. to increase the efficacy of expression or replication of the nucleic acid, or to provide additional stability or resistance to degradation.
  • the sequence of the nucleic acid molecule may be codon optimized for expression in a desired host, such as a mammalian (e.g. human) cell. Such modification with respect to codon usage may increase translation efficacy and half-life of the nucleic acid.
  • a poly A tail (e.g., of about 30 adenosine residues or more) may be attached to the 3' end of the RNA to increase its half-life.
  • the 5' end of the RNA may be capped with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which can be incorporated during RNA synthesis or can be enzymatically engineered after RNA transcription (e.g., by using Vaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase, guanylyl- transferase and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures).
  • VCE Vaccinia Virus Capping Enzyme
  • Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule.
  • the 5' cap of the RNA molecule may be further modified by a 2 '-O-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2 '-O] N), which may further increases translation efficacy.
  • nucleic acids may comprise one or more nucleotide analogs or modified nucleotides.
  • nucleotide analog or “modified nucleotide” refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions) in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U)), adenine (A) or guanine (G)).
  • a nucleotide analog can contain further chemical modifications in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analog, or open-chain sugar analog), or the phosphate.
  • ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analog, or open-chain sugar analog or the phosphate.
  • the preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art, e.g.
  • the invention further provides recombinant expression vectors capable of expressing a flagellin polypeptide.
  • the invention provides recombinant expression vectors comprising any of the nucleic acid molecules mentioned above.
  • the invention further provides host cells into which any of the vectors mentioned above have been introduced.
  • the disclosure further provides methods of producing the flagellin polypeptides as described herein by culturing the host cells under conditions permitting production of the flagellin polypeptides, and recovering the flagellin polypeptides so produced.
  • composition as disclosed herein comprising a nucleic acid sequence which encodes a flagellin polypeptides may be a nucleic acid-based therapeutic.
  • the nucleic acid may, for example, be RNA (i.e. an RNA-based therapeutic) or DNA (i.e. a DNA-based therapeutic, such as a plasmid DNA therapeutic).
  • the nucleic acid-based therapeutic is an RNA-based therapeutic.
  • the RNA-based therapeutic comprises a self-replicating RNA molecule.
  • the self-replicating RNA molecule may be an alphavirus-derived RNA replicon.
  • Self-replicating RNA molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest.
  • a self-replicating RNA molecule is typically a + strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs.
  • These daughter RNAs, as well as collinear subgenomic transcripts may be translated themselves to provide in situ expression of an encoded antigen (i.e.
  • a flagellin polypeptide may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen.
  • the overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded antigen becomes a major polypeptide product of the cells.
  • One suitable system for achieving self-replication in this manner is to use an alphavirus-based replicon.
  • These replicons are + stranded RNAs which lead to translation of a replicase (or replicase-transcriptase) after delivery to a cell.
  • the replicase is translated as a polyprotein which auto cleaves to provide a replication complex which creates genomic - strand copies of the + strand delivered RNA.
  • These - strand transcripts can themselves be transcribed to give further copies of the + stranded parent RNA and also to give a subgenomic transcript which encodes the antigen. Translation of the subgenomic transcript thus leads to in situ expression of the antigen by the infected cell.
  • Suitable alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc.
  • Mutant or wild-type virus sequences can be used e.g. the attenuated TC83 mutant of VEEV has been used in replicons.
  • the self-replicating RNA molecule described herein encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) a flagellin polypeptide.
  • the polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsP1, nsP2, nsP3 and nsP4.
  • the nucleic acid-based therapeutic may comprise a viral or a non-viral delivery system.
  • the delivery system (also referred to herein as a delivery vehicle) may have adjuvant effects which enhance the immunogenicity of the encoded flagellin polypeptide.
  • the nucleic acid molecule may be encapsulated in liposomes, non-toxic biodegradable polymeric microparticles or viral replicon particles (VRPs), or complexed with particles of a cationic oil-in-water emulsion.
  • the nucleic acid-based therapeutic comprises a cationic nano-emulsion (CNE) delivery system or a lipid nanoparticle (LNP) delivery system.
  • CNE cationic nano-emulsion
  • LNP lipid nanoparticle
  • the nucleic acid-based therapeutic may comprise viral replicon particles.
  • the nucleic acid-based therapeutic may comprise a naked nucleic acid, such as naked RNA (e.g. mRNA), but delivery via LNPs is preferred.
  • the nucleic acid-based therapeutic comprises a cationic nano-emulsion (CNE) delivery system.
  • CNE delivery systems and methods for their preparation are described in the art.
  • the nucleic acid molecule e.g. RNA
  • Cationic oil-in-water emulsions can be used to deliver negatively charged molecules, such as an RNA molecule to cells.
  • the emulsion particles comprise an oil core and a cationic lipid.
  • the cationic lipid can interact with the negatively charged molecule thereby anchoring the molecule to the emulsion particles. Further details of useful CNEs can be found in the art.
  • an RNA molecule encoding a flagellin polypeptide may be complexed with a particle of a cationic oil-in-water emulsion.
  • the particles typically comprise an oil core (e.g. a plant oil or squalene) that is in liquid phase at 25C, a cationic lipid (e.g. phospholipid) and, optionally, a surfactant (e.g. sorbitan trioleate, polysorbate 80); polyethylene glycol can also be included.
  • the CNE comprises squalene and a cationic lipid, such as 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP).
  • DOTAP 1,2-dioleoyloxy-3-(trimethylammonio)propane
  • the delivery system is a non-viral delivery system, such as CNE, and the nucleic acid-based therapeutic comprises a self-replicating RNA (mRNA). This may be particularly effective in eliciting humoral and cellular immune responses. Advantages also include the absence of a limiting anti-vector immune response and a lack of risk of genomic integration.
  • LNPs are non virion liposome particles in which a nucleic acid molecule (e.g. RNA) can be encapsulated.
  • the particles can include some external RNA (e.g. on the surface of the particles), but at least half of the RNA (and ideally all of it) is encapsulated.
  • Liposomal particles can, for example, be formed of a mixture of zwitterionic, cationic and anionic lipids which can be saturated or unsaturated, for example; DSPC (zwitterionic, saturated), DlinDMA (cationic, unsaturated), and/or DMG (anionic, saturated).
  • Preferred LNPs for use with the invention include an amphiphilic lipid which can form liposomes, optionally in combination with at least one cationic lipid (such as DOTAP, DSDMA, DODMA, DLinDMA, DLenDMA, etc.).
  • DOTAP DOTAP
  • DSDMA DODMA
  • DLinDMA DLenDMA
  • cholesterol a cationic lipid
  • Other useful LNPs are disclosed in the art.
  • the LNPs are RV01 liposomes.
  • TLR signalling pathways culminate in the activation of the transcription factor nuclear factor-kappaB (NF-kB).
  • NF-kB controls the expression of an array of inflammatory cytokine genes, including TNF- ⁇ .
  • Flagellin induces the dimerization of TLR5, which subsequently recruits MyD88 and activates protein kinases, including IRAK1, IRAK2, IRAK4 and IRAK-M.
  • the activation of these kinases leads to the nuclear localization of NF-kB, which is a proinflammatory cytokine [44].
  • compositions for use in the invention lead to an increase in expression of NF- ⁇ B. Since administration of the compositions for use in the invention increase the expression of the proinflammatory cytokine NF- ⁇ B, compositions for use in the invention may be useful in stimulating the immune response. In addition, compositions for use in the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and/or diseases treatable by an increased immune response. In one embodiment, the compositions of the invention are for use as an immune stimulant by increasing the level and/or activity of NF- ⁇ B. In one embodiment, the compositions of the invention are for use in treating diseases characterised by reduced immune activation by increasing the level and/or activity of NF- ⁇ B. In one embodiment, the compositions of the invention are for use in treating diseases treatable by an increased immune response by increasing the level and/or activity of NF- ⁇ B.
  • compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in expression and/or activation of NF- ⁇ B.
  • the compositions of the invention are for use in treating diseases characterised by a decrease in expression and/or activation of NF- ⁇ B
  • compositions of the invention are for use in treating infectious diseases, allergies and/or tumours. In one embodiment, the compositions of the invention are for use in treating infectious diseases, allergies and/or tumours by increasing the level and/or activity of NF- ⁇ B.
  • TLRs Toll-like receptors
  • PAMPs pathogen-associated molecular patterns
  • TLRs sense bacterial cell wall components and TLR5 is known to recognise bacterial flagellin.
  • Flagellin is the only known ligand for TLR5, a receptor protein expressed on the surface of a range of human cells, including epithelial cells, endothelial cells, macrophages, dendritic cells (DCs) and T cells [45-47].
  • Activation of TLR5 by bacterial flagellin leads to the activation of a variety of proinflammatory and immune response genes [15].
  • TLR5 Activation of TLR5 by S. typhimurium flagellin is known to suppress cell proliferation and tumour growth [48].
  • a S. typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) has been shown to effectively suppress tumour growth and metastasis in mouse models and prolonged survival, through a two-step activation of the TLR4 and TLR5 signalling pathways [49].
  • the efficacy of flagellin polypeptides from other species to treat cancer is unknown. Assaying the ability of a compound to activate a TLR5 response shows that the compound may be effective at treating cancer.
  • compositions comprising Enterococcus gallinarum strains, and in particular the strain MRx0518, are effective at treating and preventing cancer [50].
  • the inventors have surprisingly shown that flagellin polypeptides from the species Enterococcus gallinarum are able to stimulate a strong TLR5 response, which may contribute to the anti-cancer activity of Enterococcus strains.
  • Flagellin polypeptides from Enterococcus gallinarum have large sequence dissimilarity with flagellin polypeptides from S . typhimurium.
  • the examples show that flagellin polypeptides from the species Enterococcus gallinarum are able to produce a stronger TLR5 response than flagellin polypeptides from S . typhimurium.
  • flagellin polypeptides from the genus Enterococcus, and in particular the species Enterococcus gallinarum are useful in therapy, and particularly effective in treating and preventing cancer.
  • TLR5 has been shown to be expressed by a number of different cancers, including breast, colorectal and gastric cancer.
  • the examples show that flagellin polypeptides from the genus Enterococcus are able to produce a strong TLR5 response. Therefore, in certain embodiments the invention provides flagellin polypeptides from the genus Enterococcus for use in treating or preventing breast cancer, colorectal cancer or gastric cancer. In certain embodiments, the invention provides compositions comprising flagellin polypeptides from the genus Enterococcus for use in treating or preventing breast, colorectal or gastric cancer.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in breast, colorectal or gastric cancer.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating breast cancer.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating colorectal cancer.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating gastric cancer.
  • the invention provides compositions comprising flagellin polypeptides from the genus Enterococcus for use in treating or preventing TLR5 associated cancers.
  • the invention provides flagellin polypeptides from the genus Enterococcus for use in treating or preventing cancer.
  • the invention also provides flagellin polypeptides the species Enterococcus gallinarum for use in treating or preventing cancer. More preferably, the invention provides flagellin polypeptide from the strain MRx0518, in particular with the sequence SEQ ID NO: 1, for use in treating or preventing cancer.
  • the invention also provides compositions comprising the flagellin polypeptides described herein for use in therapy.
  • the invention also provides compositions comprising a polynucleotide sequence encoding a flagellin polypeptide as described herein for use in therapy.
  • the invention also provides a host cell that expresses a flagellin polypeptide as described herein for use in therapy.
  • the invention also provides compositions comprising a host cell, wherein the host cell comprises a polynucleotide sequence that encodes a flagellin polypeptide as described herein for use in therapy.
  • treatment with the compositions for use in the invention results in a reduction in tumour size or a reduction in tumour growth.
  • the compositions of the invention are for use in reducing tumour size or reducing tumour growth.
  • the compositions for use in the invention may be effective for reducing tumour size or growth.
  • the compositions of the invention are for use in treating patients with solid tumours.
  • the compositions of the invention are for use in reducing or preventing angiogenesis in the treatment of cancer.
  • the compositions for use in the invention may have an effect on the immune or inflammatory systems, which have central roles in angiogenesis.
  • the compositions of the invention are for use in preventing metastasis.
  • the compositions of the invention are for use in treating or preventing breast cancer.
  • Reference [50] demonstrates that Enterococcus strains have a potent effect against breast cancer, so following the data in the present application, flagellin polypeptides described herein may be particularly effective against breast cancer.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of breast cancer.
  • the cancer is mammary carcinoma.
  • the cancer is stage IV breast cancer.
  • compositions of the invention are for use in treating or preventing lung cancer.
  • Reference [50] demonstrates that Enterococcus strains have a potent effect against lung cancer, so following the data in the present application, flagellin polypeptides described herein may be particularly effective against lung cancer.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of lung cancer.
  • the cancer is lung carcinoma.
  • compositions of the invention are for use in treating or preventing liver cancer.
  • Reference [50] demonstrates that Enterococcus strains have a potent effect against liver cancer, so following the data in the present application, flagellin polypeptides described herein may be particularly effective against liver cancer.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of liver cancer.
  • the cancer is hepatoma (hepatocellular carcinoma).
  • compositions of the invention are for use in treating or preventing colorectal cancer.
  • Reference [50] demonstrates that Enterococcus strains have a potent effect against colorectal cancer, so following the data in the present application, flagellin polypeptides described herein may be particularly effective against colorectal cancer.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of colorectal cancer.
  • the cancer is colorectal adenocarcinoma.
  • the compositions of the invention are for use in treating or preventing colon cancer.
  • Reference [50] demonstrates that Enterococcus strains have a potent effect against colon cancer, so following the data in the present application, flagellin polypeptides described herein may be particularly effective against colon cancer.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of colon cancer.
  • the cancer is colon adenocarcinoma.
  • the compositions of the invention are for use in treating or preventing rectal cancer.
  • Reference [50] demonstrates that Enterococcus strains have a potent effect against rectal cancer, so following the data in the present application, flagellin polypeptides described herein may be particularly effective against rectal cancer.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of rectal cancer.
  • the cancer is rectal adenocarcinoma.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating rectal cancer.
  • the compositions of the invention are for use in treating or preventing gastric cancer.
  • Gastric carcinomas have been show to express high levels of TLR5 and that treatment with flagellin elicits a potent anti-tumour activity [51].
  • the examples show that the flagellin polypeptides described herein are able to produce a strong TLR5 response, therefore flagellin polypeptides as described herein may be particularly effective against gastric cancer.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of gastric cancer.
  • the cancer is gastric adenocarcinoma.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating gastric cancer.
  • the compositions of the invention are for use in treating or preventing melanoma.
  • Flagellin from S. typhimurium in combination with the major histocompatibility complex class II-restricted P10 peptide, has been shown to reduce the number of lung metastasis in a murine melanoma model [52].
  • the examples show that the flagellin polypeptides as described herein are able to produce a stronger TLR5 response than S. typhimurium, therefore flagellin polypeptides as described herein may be particularly effective against melanoma.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of melanoma.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating melanoma.
  • the compositions of the invention are for use in treating or preventing neuroblastoma.
  • Activation of TLR5 by S. typhimurium flagellin is known to suppress cell proliferation and tumour growth [53].
  • the examples show that the flagellin polypeptides as described herein are able to produce a stronger TLR5 response than S. typhimurium, therefore flagellin polypeptides may be particularly effective against neuroblastoma.
  • the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of neuroblastoma.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating neuroblastoma.
  • the cancer is of the intestine. In some embodiments, the cancer is of a part of the body which is not the intestine. In some embodiments, the cancer is not cancer of the intestine. In some embodiments, the cancer is not colorectal cancer. In some embodiments, the cancer is not cancer of the small intestine. In some embodiments, the treating or preventing occurs at a site other than at the intestine. In some embodiments, the treating or preventing occurs at the intestine and also at a site other than at the intestine.
  • TLR5 has been detected in haematological cancer cell lines, including multiple myeloma [54] and acute myeloid leukaemia [55].
  • the inventors have demonstrated in the examples that flagellin polypeptides from the genus Enterococcus are particularly effective at activating a strong TLR5 response.
  • Flagellin polypeptides from the genus Enterococcus may therefore be useful at treating or preventing haematological cancers, such as multiple myeloma and acute myeloid leukaemia, which express TLR5.
  • the invention provides compositions comprising flagellin polypeptides from the genus Enterococcus for use in treating or preventing haematological malignancies, such as multiple myeloma, acute and chronic leukemias such as acute myeloid leukaemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia and acute monocytic leukemia, lymphomas, such as Hodgkin's lymphomas and Non-Hodgkin's lymphomas, and myelodysplastic syndromes.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating acute myeloid leukaemia.
  • the invention provides compositions comprising flagellin polypeptides from the genus Enterococcus for use in treating or preventing multiple myeloma. In certain embodiments, the invention provides compositions comprising flagellin polypeptides from the genus Enterococcus for use in treating or preventing acute myeloid leukaemia. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating multiple myeloma.
  • TLR5 agonists have been tested in a wide array of tumour models and clinical trials [56].
  • the TLR5 agonist CBLB502 has been shown to promote tumour clearance in mice models of T and B cell lymphomas [57].
  • Systemic administration of the TLR5 agonist entolimod suppressed liver metastasis of colorectal cells in mice models.
  • the liver shows the strongest TLR5 activation response following the administration of the TLR5 agonist entolimod [58].
  • flagellin polypeptides from the genus Enterococcus act as strong TLR5 agonists.
  • flagellin polypeptides from the genus Enterococcus may be particularly effective at treating or preventing T and B cell lymphomas, colorectal and liver cancer.
  • the compositions of the invention are for use in treating or preventing T and B cell lymphomas.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating T and B cell lymphomas.
  • the compositions for use in the invention may be useful in treating or preventing metastasis. In other embodiments, the compositions for use in the invention may be useful in treating metastatic cancer. In other embodiments, the compositions for use in the invention may be useful in treating advanced cancer. In certain embodiments, the invention provides compositions comprising flagellin polypeptides from the genus Enterococcus for use in treating, reducing or preventing metastasis. In certain embodiments, the compositions for use in the invention may decrease or prevent metastatic growth. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating or preventing metastasis.
  • the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating or preventing metastatic cancer. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum, for use in treating or preventing advanced cancer.
  • compositions of the invention are for use in treating or preventing carcinoma.
  • Vibrio vulnificus flagellin B has been shown to effectively suppress tumour growth through a two-step activation of the TLR4 and TLR5 signalling pathways.
  • the flagellin polypeptides can activate the TLR4 and TLR5 signalling pathways. In other embodiments the flagellin polypeptides only activate the TLR5 signalling pathway.
  • the flagellin polypeptides can activate NT ⁇ B signalling pathways.
  • host cells for use in the invention can activate the TLR9 signalling pathways.
  • Reference [59] shows that synergy between flagellin and the TLR9 agonist CpG-containing oligodeoxynucleotides leads to tumour suppression.
  • the host cells for use in the invention can activate the TLR5 and TLR9 signalling pathways.
  • the composition for use in the invention comprises CpG-containing oligodeoxynucleotides or is administered in combination with CpG-containing oligodeoxynucleotides.
  • compositions for use in the invention may be mediated by a pro-inflammatory mechanism.
  • Activation of TLR5 by flagellin initiates a proinflammatory signal cascade.
  • Reference [59] shows that the interaction of S. typhimurium flagellin with highly immunogenic tumours induces a Th1 response and suppression of Tregs, resulting in the inhibition of tumour growth. In contrast, the growth rate of weakly immunogenic tumours was not affected by flagellin administration. Therefore, in certain embodiments, the flagellin polypeptides and compositions of the invention are for use in treating immunogenic tumours, in particular highly immunogenic tumours. In certain embodiments, the compositions of the invention are for use in promoting inflammation in the treatment of cancer.
  • compositions of the invention are for use in promoting Th1 inflammation in the treatment of cancer.
  • Th1 cells produce IFN ⁇ and have potent anti-cancer effects [60].
  • the compositions of the invention are for use in treating an early-stage cancer, such as a cancer that has not metastasized, or a stage 0 or stage 1 cancer. Promoting inflammation may be more effective against early-stage cancers [60].
  • Reference [59] shows that administration of flagellin after tumour transplantation significantly inhibited growth of antigenic tumours.
  • the differing effects of flagellin on tumour growth are correlated with the immune response that is induced.
  • Flagellin administration after transplantation was associated with an increased IFN-y:IL-4 ratio and the decreased frequency of CD4+CD25+ T regulatory cells. Therefore, in certain embodiments, the flagellin polypeptides and compositions for use in the invention increase the IFN-y:IL-4 ratio. In certain embodiments, the flagellin polypeptides and compositions for use in the invention decrease the frequency of CD4+CD25+ T regulatory cells.
  • the flagellin polypeptides and compositions for use in the invention do not decrease the IFN- ⁇ :IL-4 ratio and increase CD4+CD25+ T cell frequency.
  • Inflammation can have a cancer-suppressive effect [60] and pro-inflammatory cytokines such as TNF ⁇ are being investigated as cancer therapies [61].
  • the up-regulation of genes such as TNF may indicate that the compositions for use in the invention may be useful for treating cancer via a similar mechanism.
  • the up-regulation of CXCR3 ligands (CXCL9, CXCL10) and IFN ⁇ -inducible genes (IL-32) may indicate that the compositions for use in the invention elicit an IFN ⁇ -type response.
  • IFN ⁇ is a potent macrophage-activating factor that can stimulate tumirocidal activity [62], and CXCL9 and CXCL10, for example, also have anti-cancer effects [63-65].
  • the compositions of the invention are for use in promoting inflammation to enhance the effect of a second anti-cancer agent.
  • the treatment or prevention of cancer comprises increasing the level of expression of one or more cytokines.
  • the treatment or prevention of cancer comprises increasing the level of expression of one or more of IL-1 ⁇ , IL-6 and TNF- ⁇ , for example, IL-1 ⁇ and IL-6, IL-1 ⁇ and TNF- ⁇ , IL-6 and TNF- ⁇ or all three of IL-1 ⁇ , IL-6 and TNF- ⁇ .
  • Increases in levels of expression of any of IL-1 ⁇ , IL-6 and TNF- ⁇ are known to be indicative of efficacy in treatment of cancer.
  • compositions of the invention are for use in treating a patient that has previously received chemotherapy. In certain embodiments, the compositions of the invention are for use in treating a patient that has not tolerated a chemotherapy treatment.
  • the compositions for use in the invention may be particularly suitable for such patients.
  • compositions for use in the invention are for preventing relapse.
  • the compositions for use in the invention may be suitable for long-term administration.
  • the compositions of the invention are for use in preventing progression of cancer.
  • compositions of the invention are for use in treating non-small-cell lung carcinoma. In certain embodiments, the compositions of the invention are for use in treating small-cell lung carcinoma. In certain embodiments, the compositions of the invention are for use in treating squamous-cell carcinoma. In certain embodiments, the compositions of the invention are for use in treating adenocarcinoma. In certain embodiments, the compositions of the invention are for use in treating glandular tumors, carcinoid tumors, or undifferentiated carcinomas.
  • compositions of the invention are for use in treating hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancer resulting from a viral infection.
  • compositions of the invention are for use in treating invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma.
  • compositions of the invention are for use in treating or preventing acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esoph
  • compositions for use in the invention may be particularly effective when used in combination with further therapeutic agents.
  • the immune-modulatory effects of the compositions for use in the invention may be effective when combined with more direct anti-cancer agents. Therefore, in certain embodiments, the invention provides a composition comprising a flagellin polypeptide from the genus Enterococcus (or encoding polynucleotide or expressing host cell) and an anticancer agent.
  • the anticancer agent is an immune checkpoint inhibitor, a targeted antibody immunotherapy, a CAR-T cell therapy, an oncolytic virus, or a cytostatic drug.
  • the anti-cancer agent is Keytruda (pembrolizumab, Merck).
  • the composition comprises an anti-cancer agent selected from the group consisting of: Yervoy (ipilimumab, BMS); Opdivo (nivolumab, BMS); MEDI4736 (AZ/MedImmune); MPDL3280A (Roche/Genentech); Tremelimumab (AZ/MedImmune); CT-011 (pidilizumab, CureTech); BMS-986015 (lirilumab, BMS); MEDI0680 (AZ/MedImmune); MSB-0010718C (Merck); PF-05082566 (Pfizer); MEDI6469 (AZ/MedImmune); BMS-986016 (BMS); BMS-663513 (urelumab, BMS); IMP321 (Prima Biomed); LAG525 (Novartis); ARGX-110 (arGEN-X); PF-05082466 (Pfizer); CDX-1127 (varlilumab
  • flagellin polypeptides are administered in combination (contemporaneously or sequentially) with CpG-containing oligodeoxynucleotides, which have shown useful effects with other flagellins [59].
  • a flagellin polypeptide from the genus Enterococcus is the only therapeutically active agent(s) in a composition for use in the invention.
  • the invention provides a flagellin polypeptide from the genus Enterococcus, preferably a flagellin polypeptide from the species Enterococcus gallinarum, preferably a flagellin polypeptide from the strain MRx0518, for use in preventing or treating symptoms associated with toxic chemical, pathogen or radiation exposure.
  • the flagellin may be administered to subjects at risk of toxic chemical, pathogen or radiation exposure, or subjects recently exposed to toxic chemicals, pathogens or radiation, for example subjects exposed 1, 2, 3, 4, or 5 hours previously.
  • Flagellin stimulates host defence in a variety of organisms, including mammals, insects, and plants.
  • flagellin is a potent activator of innate immune signalling pathways in epithelial cells but has generally observed to be a poor activator of hemopoietic cells, such as macrophages and dendritic cells.
  • S. typhimurium flagellin has been observed to not stimulate significant levels of "master" proinflammatory cytokines, such as TNF- ⁇ , that mediate the adverse effects associated with LPS.
  • S. typhimurium flagellin has been observed to activate TLR5 mediated innate immunity in mice, protecting the mice against chemical, pathogen or radiation exposure [66].
  • S. typhimurium flagellin has been shown to protect against radiation exposure via a mechanism requiring TLR5 [66]. Optimal protection was observed when flagellin was administered prophylactically 2 hours before radiation, but a significant degree of protection was still observed if the flagellin was administered up to 4 h following irradiation.
  • the examples show that flagellin polypeptides from the genus Enterococcus are able to stimulate a stronger TLR5 response than S . typhimurium.
  • the inventors have shown that flagellin polypeptides from the genus Enterococcus, and in particular the species Enterococcus gallinarum may be useful as radioprotective agents.
  • the invention provides flagellin polypeptides from the genus Enterococcus for use as radioprotective agents.
  • the invention also provides flagellin polypeptides the species Enterococcus gallinarum for use as radioprotective agents. More preferably, the invention provides flagellin polypeptide from the strain MRx0518, in particular with the sequence SEQ ID NO:1, for use as radioprotective agents.
  • TLR5 agonist Entolimod Treatment with the TLR5 agonist Entolimod has also been shown to reduce radiation-induced damage in all three of the major types of radiosensitive tissues: hemato-poietic, gastrointestinal and skin. It has also been shown to mitigate radiation-induced epithelial damage in a mouse model of head and neck radiation [67].
  • the examples show that flagellin polypeptides from the genus Enterococcus act as a potent TLR5 agonist.
  • flagellin polypeptides from the genus Enterococcus, and in particular the species Enterococcus gallinarum may be useful at reducing radiation-induced damage.
  • compositions comprising flagellin polypeptides from the genus Enterococcus are for use in reducing radiation-induced damage in hemato-poietic, gastrointestinal or skin tissues. In certain embodiments, compositions comprising flagellin polypeptides from the genus Enterococcus are for use in reducing radiation-induced epithelial damage. In a preferred embodiment, the invention provides a composition comprising flagellin polypeptides from the species Enterococcus gallinarum for use in reducing radiation-induced damage.
  • the composition may be administered, preferably orally, prior to radiotherapy. In certain embodiments, the composition may be administered, preferably orally, shortly after radiotherapy. In certain embodiments, the compositions are to be administered to a patient that has recently undergone radiotherapy, or that is scheduled to undergo radiotherapy.
  • the flagellin polypeptides from the genus Enterococcus are administered before the irradiation exposure. In other embodiments, the flagellin polypeptides from the genus Enterococcus are administered after the radiation exposure, preferably within 4 hours following exposure.
  • S. typhimurium flagellin has been shown stimulate immune responses to bacterial infection.
  • Reference [68] shows that the prophylactic intranasal administration of S. typhimurium flagellin with an antibiotic protects mice against respiratory Streptococcus pneumoniae bacteria, while reference [66] shows that oral administration of S. typhimurium flagellin 2 hours prior to infection with S. typhimurium reduced mortality.
  • Flagellin induced these responses by activating the TLR5 innate immune response.
  • the examples show that flagellin polypeptides from the genus Enterococcus are able to stimulate a stronger TLR5 response than S. typhimurium.
  • the inventors have shown that flagellin polypeptides from the genus Enterococcus, and in particular the species Enterococcus gallinarum can be useful in the treatment or prevention of bacterial infections.
  • the invention provides flagellin polypeptides from the genus Enterococcus for use in treating or preventing bacterial infections.
  • the invention also provides flagellin polypeptides the species Enterococcus gallinarum for use in treating or preventing bacterial infections. More preferably, the invention provides flagellin polypeptide from the strain MRx0518, in particular with the sequence SEQ ID NO: 1, for use in treating or preventing bacterial infections.
  • the flagellin polypeptides from the genus Enterococcus are used in combination with an antibiotic for use in treating bacterial infections. In certain embodiments, the flagellin polypeptides from the genus Enterococcus are used the only therapeutic agent in a composition for use in treating bacterial infections.
  • Flagellin has been shown to act as adjuvants in vaccines for bacterial, viral and parasitic infections, however, most of these studies used flagellin from S. typhimurium and Vibrio vulnificus [15]. The efficacy of flagellin polypeptides from other species to act as vaccine adjuvants, and in particular Enterococcus spp., is unknown.
  • Flagellin acts as an adjuvant by stimulating the innate immune response through the TLR5 receptor.
  • the inventors have surprisingly shown that flagellin polypeptides from the genus Enterococcus are able to stimulate a stronger TLR5 response than S. typhimurium.
  • flagellin polypeptides from the genus Enterococcus, and in particular the species Enterococcus gallinarum can be useful as vaccine adjuvants.
  • Use of a flagellin polypeptide as a vaccine adjuvant means that the flagellin polypeptide is used, for example, to provide protection against an infective agent or prevent infection by an infective agent, generally by enhancing the immune response to a separate antigen.
  • the invention provides flagellin polypeptides from the genus Enterococcus for use as vaccine adjuvants.
  • the invention also provides flagellin polypeptides the species Enterococcus gallinarum for use as vaccine adjuvants. More preferably, the invention provides flagellin polypeptide from strain MRx0518, in particular with the sequence SEQ ID NO:1, for use as vaccine adjuvants.
  • the disclosure also provides vaccine compositions comprising flagellin polypeptides as described herein as an adjuvant.
  • the vaccine compositions are for use in treating or preventing bacterial infections.
  • the bacterial pathogen to be treated or prevented is Y. pestis, tetanus, Streptococcus pneumoniae, Escherichia coli or M. tuberculosis.
  • the vaccine compositions are for use in treating or preventing viral infections.
  • the viral pathogen to be treated or prevented is influenza, HIV, rabies or foot and mouth virus.
  • the vaccine compositions are for use in treating or preventing parasitic infections.
  • the parasite is Plasmodium vivax, Plasmodium yoelii or Eimeria tenella.
  • the composition comprises at least one antigen from the relevant pathogen.
  • the composition comprises the flagellin as described herein fused or conjugated to at least one antigen from the relevant pathogen.
  • the invention provides a fusion polypeptide comprising a flagellin as described herein, an optional linker, and an antigen from a pathogen or parasite.
  • Flagellin is a potent activator of innate immune signalling pathways in epithelial cells, which are the first major cell type that encounter agents administered by a mucosal route.
  • S. typhimurium and Vibrio vulnificus flagellin proteins have been shown to potent adjuvant activity in mucosal vaccines against influenza, West Nile virus, Escherichia coli, Yersinia pestis, Clostridium tetani, C. jejuni, Streptococcus spp. and Plasmodium falciparum [15].
  • the efficacy of flagellin polypeptides from other species to act as mucosal vaccines adjuvants is unknown.
  • Flagellin acts as an adjuvant by stimulating the innate immune response through the TLR5 receptor.
  • the inventors have surprisingly shown that flagellin polypeptides from the genus Enterococcus are able to stimulate a stronger TLR5 response than S. typhimurium.
  • the invention provides flagellin polypeptides from the genus Enterococcus for use as a mucosal vaccine adjuvant.
  • the invention provides flagellin polypeptides from the species Enterococcus gallinarum for use as mucosal vaccine adjuvants.
  • the invention provides flagellin polypeptide from strain MRx0518, in particular with the sequence SEQ ID NO:1, for use mucosal as vaccine adjuvants.
  • flagellin polypeptides from the genus Enterococcus are for use in mucosal vaccine for use in treating or preventing bacterial infections.
  • the bacterial infection is Escherichia coli, Yersinia pestis, Clostridium tetani, C. jejuni, Streptococcus spp. or Plasmodium falciparum.
  • flagellin polypeptides from the genus Enterococcus are for use in mucosal vaccines for use in treating or preventing viral infections.
  • the viral infection is influenza or the West Nile virus.
  • compositions for use in the invention may lead to an increase in expression of Tumour Necrosis Factor alpha (TNF- ⁇ ).
  • TNF- ⁇ Tumour Necrosis Factor alpha
  • TNF- ⁇ is known to be important for vaccine responses.
  • TNF- ⁇ has been shown to be required for an efficient vaccine response in a flu vaccination of the elderly population [69]. Since administration of the compositions for use in the invention may increase TNF- ⁇ expression, compositions for use in the invention may be useful as a vaccine adjuvant.
  • the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of TNF- ⁇ .
  • the compositions of the invention are for use as a vaccine adjuvant.
  • the compositions of the invention are for use as a vaccine adjuvant in influenza therapy.
  • the compositions of the invention are for use in enhancing an immune response against an antigen.
  • the invention provides a composition to be administered in combination with an antigen.
  • the flagellin for use in the invention may be fused or conjugated to the antigen.
  • the compositions for use in the invention are for administration to a patient shortly prior to or after vaccination.
  • Enterococcus gallinarum and in particular strain MRX518 is flagellated and flagellins can be TLR5 agonists.
  • TLR agonists are in development as vaccine adjuvants across a range of antigen types, particularly in the elderly population [70].
  • MRX518 is a TLR5 agonist. Therefore, the compositions for use in the invention may be useful as vaccine adjuvants, in particular for vaccine administered to elderly patients (e.g. over 40, 50, 60, 70 or 80 years of age), who may have reduced immune system activity.
  • TLR5 signalling also plays a key role in age-associated innate immune responses [71].
  • the compositions are for use in enhancing an innate immune response.
  • TLR5 agonists are in development as vaccine adjuvants, these are all from known pathogens and/or synthetic.
  • the compositions for use in the invention comprise commensal bacteria.
  • compositions for use in the invention may lead to an increase in expression of IL-6.
  • Increased 11-6 expression has been associated with vaccine responses for many diseases.
  • IL-6 was produced by CD14+CD16- inflammatory monocytes after adults were administered an influenza vaccine [72], and higher levels of IL-6 were associated with achieving a vaccine response to an influenza vaccine [73].
  • 11-6 was produced after injection of the AS03 adjuvant system [74] and downregulation of IL-6 in mice was shown to reduce the helper T cell response after administration of a tuberculosis vaccine [75].Since administration of the compositions for use in the invention may increase IL-6 expression, compositions for use in the invention may be useful as a vaccine adjuvant.
  • compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of IL-6. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in tuberculosis therapy.
  • compositions for use in the invention may be useful as a vaccine adjuvant.
  • compositions for use in the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of TNF- ⁇ . In one embodiment, the compositions for use in the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of IL-6. In a particular embodiment, the compositions for use in the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of TNF- ⁇ and IL-6. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in HIV therapy. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in chlamydia therapy.
  • compositions for use in the invention may lead to an increase in expression of IL-1 ⁇ .
  • Li et al. [78] showed that the adjuvant aluminium hydroxide activated the secretion of IL-1 ⁇ , and suggested that IL- ⁇ itself can act as an adjuvant. Since administration of the compositions for use in the invention may increase IL-1 ⁇ expression, compositions for use in the invention may be useful as a vaccine adjuvant. The examples show that administration of the compositions for use in the invention can increase the ratio of CD8+ T cells to Tregs.
  • compositions for use in the invention may be useful as a vaccine adjuvant.
  • compositions of the invention are for use as a vaccine adjuvant.
  • the compositions of the invention are for use as a vaccine adjuvant by increasing the ratio of CD8+ T cells to Tregs.
  • compositions for use in the invention may lead to an increase in expression or levels of CXCR3 ligands CXCL9 and CXCL10.
  • Known adjuvants such as ASO3, CpG, GLA-SE, ⁇ GalCer all increase CXCL9 and 10 [80,81], which suggests the compositions for use in the invention will be effective as adjuvants.
  • CXCL9 and 10 are associated with IFN ⁇ /Th1 responses and promote antibody responses [82].
  • the compositions of the invention are for use in promoting an antibody response against an antigen, in particular a pathogenic or cancer antigen.
  • CXCL9 is a more sensitive measure than IFN-y of vaccine induced T-cell responses in volunteers receiving investigated malaria vaccines [83].
  • compositions of the invention are for use in promoting an T-cell response against an antigen, in particular a pathogenic or cancer antigen.
  • compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of CXCL9 and CXCL10.
  • the compositions are for use in protecting against malaria.
  • compositions for use in the invention can lead to an increase in expression or levels of IL-12p70. This effect has been associated with vaccine adjuvant efficiency and IL-12 has been proposed as an adjuvant itself [84], which suggests the compositions for use in the invention will be effective as adjuvants.
  • the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of IL-12p70.
  • compositions for use in the invention when used as a vaccine adjuvant, will be administered on their own to provide an adjuvant effect for an antigen that has been separately administered to the patient.
  • the composition for use in the invention is administered orally, whilst the antigen is injected parenterally.
  • the flagellin for use in the invention may be fused or conjugated to the antigen.
  • compositions for use in the invention may be used for enhancing an immune response to any useful antigen.
  • exemplary antigens for use with the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens.
  • the invention is particularly useful for vaccines against influenza virus, HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cytomegalovirus, Staphylococcus aureus, chlamydia, SARS coronavirus, varicella zoster virus, Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus, human papillomavirus, etc.
  • the flagellin for use in the invention is fused or conjugated to one or more of these antigens.
  • antigens for use with the flagellins for use in the invention include glycoprotein and lipoglycan antigens.
  • the antigen is an archaea antigen.
  • Exemplary tumour-associated antigens include melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens and oncoproteins.
  • MAGE melanoma antigen E
  • CEA Carcinoembryonic antigen
  • MUC-1 MUC-1
  • HER2 sialyl-Tn
  • STn sialyl-Tn
  • hTERT human telomerase reverse transcriptase
  • WT1 Wilms tumour gene
  • CA-125 CA-125
  • PSA prostate-specific anti
  • Flagellins for use in the invention may also be useful for enhancing the response to vaccines against non-communicable diseases such as Alzheimer's Disease and other neurodegenerative disorders, in which case the antigen for use with the invention may be amyloid-beta or Tau. Other such antigens for non-communicable diseases include PCSK9 (for the treatment of elevated cholesterol). Flagellins for use in the invention may also be useful for enhancing the response to vaccines against habit forming substances, for example nicotine, alcohol or opiates.
  • the immune response raised by these methods and uses will generally include an antibody response, preferably a protective antibody response.
  • compositions for use in the invention can lead to immune stimulation. Since administration of the compositions for use in the invention may have an immunostimulatory effect, compositions for use in the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and diseases treatable by an increased immune response.
  • the compositions of the invention are for use in stimulating the immune system.
  • the compositions of the invention are for use in treating disease by stimulating the immune system.
  • the compositions of the invention are for use in promoting an immune response.
  • compositions for use in the invention may be useful in the treatment of diseases characterised by an increase in the percentage of Tregs in a cell population.
  • the compositions for use in the invention may be useful for treating or preventing diseases characterised by an increase in the percentage of Tregs in a cell population.
  • the compositions for use in the invention may be useful for treating or preventing diseases characterised by an increase in the percentage of CD4+CD25+CD127- cells in a cell population.
  • the compositions of the invention are for use in treating or preventing diseases by decreasing the percentage of Tregs in cell populations.
  • compositions of the invention are for use in reducing suppression of the immune response by Tregs.
  • compositions of the invention are for use in stimulating the immune response by the selective reduction of Tregs.
  • compositions of the invention are for use in immunostimulation, wherein the compositions reduce the number or percentage of Tregs.
  • compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in the ratio of CD8/Treg and/or activated CD8/Treg cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the ratio of CD8/Treg cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the ratio of activated CD8/Treg cells.
  • compositions of the invention are for use in stimulating the immune response by increasing the ratio of CD8/Treg cells.
  • compositions of the invention are for use in stimulating the immune response by increasing the ratio of activated CD8/Treg cells.
  • compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of B cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of B cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD19+CD3- cells.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of B cells in cell populations, wherein the increase in number or percentage of B cells results in immune stimulation.
  • compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of B cells.
  • compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of CD8 T-cytotoxic cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD8 T-cytotoxic cells.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of CD8 T-cytotoxic cells in cell populations, wherein the increase in number or percentage of CD8 T-cytotoxic cells results in immune stimulation.
  • compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of CD8 T-cytotoxic cells.
  • compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of CD8+ activated cells.
  • the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CD8+ activated cells.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of CD8+ activated cells in cell populations, wherein the increase in number or percentage of CD8+ activated cells results in immune stimulation.
  • compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of CD8+ activated cells.
  • compositions for use in the invention may lead to an increase in expression of pro-inflammatory molecules, such as pro-inflammatory cytokines.
  • pro-inflammatory molecules such as pro-inflammatory cytokines.
  • pro-inflammatory molecules that may show an increase in expression levels upon administration of compositions for use in the invention include IL-8, IL-12p70, IL-23, TNF- ⁇ , IL-1 ⁇ , and IL-6. Since administration of the compositions for use in the invention may increase the expression of pro-inflammatory molecules, compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in expression of pro-inflammatory molecules, such as pro-inflammatory cytokines.
  • compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of pro-inflammatory molecules, in particular diseases characterised by a decrease in the expression and/or activity of pro-inflammatory cytokines.
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-8, IL-12p70, IL-23, TNF- ⁇ , IL-1 ⁇ ,- and/or IL-6.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-23, TNF- ⁇ , IL-1 ⁇ , and/or IL-6.
  • compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-8, IL-12p70, IL-23, TNF- ⁇ , IL-1 ⁇ , and/or IL-6.
  • compositions for use in the invention may lead to an increase in expression of IL-1 ⁇ .
  • IL-1 ⁇ is a pro-inflammatory cytokine [85].
  • the production and secretion of IL-1 ⁇ is regulated by the inflammasome, a protein complex which is associated with activation of the inflammatory response [86].
  • administration of the compositions for use in the invention may increase the expression of IL-1 ⁇
  • compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-1 ⁇ .
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-1 ⁇ .
  • the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-1 ⁇ .
  • compositions for use in the invention may lead to an increase in expression of IL-23.
  • IL-23 has been linked to inflammation [87,88].
  • the proposed functions of IL-23 in the immune response include promoting the proliferation of CD4+ memory T cells and promoting the secretion of IFN-y by dendritic cells (DCs) [89].
  • DCs dendritic cells
  • administration of the compositions for use in the invention may increase the expression of IL-23
  • compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-23.
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-23.
  • compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-23. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-23.
  • TNF- ⁇ Tumour Necrosis Factor alpha
  • TNF- ⁇ is a pro-inflammatory cytokine which is known to be involved in various signalling pathways to promote cell death. TNF- ⁇ initiates apoptosis by binding to its cognate receptor, TNFR-1, which leads to a cascade of cleavage events in the apoptotic pathway [90]. TNF- ⁇ can also trigger necrosis via a RIP kinase-dependent mechanism [91].
  • compositions for use in the invention may be useful in the treatment of diseases, in particular for use in treating or preventing diseases characterised by a decrease in expression of by TNF- ⁇ .
  • the compositions of the invention are for use in treating diseases characterised by decreased TNF- ⁇ expression.
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of TNF- ⁇ .
  • the compositions for use in the invention may be useful for treating or preventing diseases by increasing the expression and/or activity of TNF- ⁇ .
  • compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of TNF- ⁇ .
  • compositions for use in the invention may lead to an increase in expression of IL-6.
  • IL-6 a pro-inflammatory cytokine that is produced during inflammation, and promotes the differentiation of naive CD4+ T cells and the differentiation of CD8+ T cells into cytotoxic T cells [ 92 ]. Since administration of the compositions for use in the invention may increase the expression of IL-6, compositions for use in the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-6.
  • the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-6.
  • the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-6.
  • compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-6.
  • compositions for use in the invention may increase the expression of IL-6, so compositions for use in the invention may selectively decrease the number or percentage of Tregs by increasing the expression of IL-6.
  • compositions of the invention are for use in immunostimulation by increasing the expression of IL-6.
  • compositions of the invention are for use in immunostimulation by decreasing the number or percentage of Tregs.
  • compositions for use in the invention promote the differentiation of T-helper cells and cytotoxic T lymphocytes. Therefore, in certain embodiments, the compositions of the invention are for use in stimulating the differentiation of T-helper cells and/or cytotoxic T lymphocytes.
  • compositions for use in the invention may lead to an increase in expression of IL-6. Increased 11-6 expression has been correlated with response to CD19 CAR-T therapy of chronic lymphocyte leukaemia. An increase in serum IL-6 was associated with CAR-T cell expansion, whereas inhibition of IL-6 was associated with inhibition of CAR-T cell proliferation [94]. Since administration of the compositions for use in the invention may increase IL-6 expression, compositions for use in the invention may be useful in cell therapy, in particular CAR-T cell therapy. In one embodiment, the compositions of the invention are for use in cell therapy. In one embodiment, the compositions of the invention are for use in CAR-T cell therapy. In one embodiment, compositions of the invention are for use in the treatment of chronic lymphocyte leukaemia.
  • compositions for use in the invention may be useful in cell therapy, in particular CAR-T cell therapy.
  • compositions for use in the invention may be useful in cell therapy, in particular in enhancing the response to a cell therapy.
  • MSC Mesynchymal stem cell
  • MSC Mesynchymal stem cell
  • compositions for use in the invention may initiate neuronal differentiation in undifferentiated neuroblastoma cells, compositions for use in the invention may be useful for stem cell differentiation in stem cell transplantation therapy.
  • Hematopoietic stem cell transplantation is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. Colonisation of the gut with Enterococci ( Enterococcus gallinarum and Enterococcus casseliflavus ) prior to allogenic hematopoietic stem cell transplantation has been shown to lead to a significantly improved the 2-year survival of patients after due to decreased nonrelapse mortality [ 98 ]. Therefore, the immunomodulatory effect shown in the examples may be useful in hematopoietic stem cell transplantation therapy. In certain embodiments, the compositions for use in the invention may be useful in improving survival after hematopoietic stem cell transplantation and in particular after allogenic hematopoietic stem cell transplantation.
  • compositions for use in the invention may be useful in combination with allogenic hematopoietic stem cell transplantation.
  • the compositions for use in the invention may be effecting in boosting successful patient response to allogenic hematopoietic stem cell transplantation.
  • the compositions for use in the invention are administered prior to hematopoietic stem cell transplantation.
  • the compositions for use in the invention are for administration to a patient scheduled to receive hematopoietic stem cell transplantation.
  • the compositions for use in the invention are administered following hematopoietic stem cell transplantation.
  • the compositions for use in the invention are for administration to a patient that has received hematopoietic stem cell transplantation.
  • compositions for use in the invention may be used to prevent or delay immunosenescence.
  • compositions of the invention are for use in preventing immunosenescence.
  • compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number.
  • compositions of the invention are for use in delaying immunosenescence characterised by a decrease in B cell number.
  • compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number and a decrease in B cell number.
  • compositions of the invention are for use in delaying immunosenescence by decreasing Treg cell number. In one embodiment, compositions of the invention are for use in delaying immunosenescence by increasing B cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence by decreasing Treg cell number and increasing B cell number. In one embodiment, compositions of the invention are for use in treating diseases caused by immunosenescence. In one embodiment, compositions of the invention are for use in treating aging-related diseases by delaying and/or preventing immunosenescence.
  • compositions for use in the invention may be useful for preventing or delaying immunosenescence.
  • compositions of the invention are for use in delaying and/or preventing immunosenescence as a vaccine adjuvant.
  • compositions of the invention are for use as a vaccine adjuvant, wherein the compositions delay and/or prevent immunosenescence.
  • compositions for use in the invention are administered by injection, preferably subcutaneously or alternatively intravenously, or intraperitoneally. Further details regarding compositions suitable for administered by injection are provided in the next section.
  • the compositions for use in the invention are to be administered to the gastrointestinal tract.
  • the immune system is known to be modulated by the presence of bacteria and bacterial proteins in the gastrointestinal tract.
  • the compositions for use in the invention are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes.
  • compositions for use in the invention may be administered as a foam, as a spray or a gel.
  • compositions for use in the invention may be administered as a suppository, such as a rectal suppository, for example in the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.
  • a rectal suppository for example in the form of a theobroma oil (coa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.
  • the composition for use in the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.
  • a tube such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.
  • compositions for use in the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions for use in the invention are to be administered daily.
  • treatment according to the invention is accompanied by assessment of the patient's gut microbiota.
  • compositions for use in the invention may be administered to a patient that has been diagnosed with cancer, or that has been identified as being at risk of a cancer.
  • the compositions may also be administered as a prophylactic measure to prevent the development of cancer in a healthy patient.
  • compositions for use in the invention may be administered to a patient that has been identified as having an abnormal gut microbiota.
  • the patient may have reduced or absent colonisation by Enterococcus spp, in particular Enterococcus gallinarum.
  • compositions for use in the invention are for the treatment of humans, although they may be used to treat animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits.
  • the compositions for use in the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used.
  • composition for use in the invention comprises a flagellin polypeptide from the genus Enterococcus and in particular from the species Enterococcus gallinarum.
  • the composition for use in the invention is encapsulated to enable delivery of flagellin to the intestine.
  • Encapsulation protects the composition from degradation until delivery at the target location through, for example, rupturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule.
  • compositions may thus be pharmaceutically acceptable. They will usually include components in addition to the flagellin polypeptide (or nucleic acid) e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s).
  • compositions may be administered to a human in aqueous form.
  • the composition prior to administration, however, the composition may have been in a non-aqueous form.
  • some therapeutics are manufactured in aqueous form, then filled and distributed and administered also in aqueous form, other therapeutics are lyophilised during manufacture and are reconstituted into an aqueous form at the time of use.
  • a composition for use in the invention may be dried, such as a lyophilised formulation.
  • the composition may include preservatives such as thiomersal or 2-phenoxyethanol. It is preferred, however, that the therapeutics should be substantially free from ( i.e. less than 5 ⁇ g/ml) mercurial material e.g. thiomersal-free. Therapeutics containing no mercury are more typical. Preservative-free therapeutics are particularly favoured.
  • preservatives such as thiomersal or 2-phenoxyethanol. It is preferred, however, that the therapeutics should be substantially free from ( i.e. less than 5 ⁇ g/ml) mercurial material e.g. thiomersal-free. Therapeutics containing no mercury are more typical. Preservative-free therapeutics are particularly favoured.
  • a composition may include a temperature protective agent. Further details of such agents are provided below.
  • a physiological salt such as a sodium salt.
  • Sodium chloride (NaCl) is generally used, which may be present at between 1 and 20 mg/ml e.g. about 10+2mg/ml NaCl.
  • Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.
  • Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, more often between 240-360 mOsm/kg, and will more typically fall within the range of 290-310 mOsm/kg.
  • Compositions may include one or more buffers.
  • Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20mM range.
  • the pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.
  • the composition is typically sterile.
  • the composition is also typically non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, for example ⁇ 0.1 EU per dose.
  • the composition is often gluten free.
  • the composition may include material for a single administration, or may include material for multiple administration (i.e. a 'multidose' kit).
  • a preservative is typical in multidose arrangements.
  • the compositions may be contained in a container having an aseptic adaptor for removal of material.
  • Human protein therapeutics are typically administered in a dosage volume of about 0.5ml, although a half dose (i.e. about 0.25ml) may be administered to children.
  • compositions for use in the invention may also comprise one or more immunoregulatory agents.
  • one or more of the immunoregulatory agents include one or more adjuvants.
  • the adjuvants may include a TH1 adjuvant and/or a TH2 adjuvant, further discussed below.
  • compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition).
  • the composition may be prepared for topical administration e.g. as an ointment, cream or powder.
  • the composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured).
  • the composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • the composition may be prepared for nasal, aural or ocular administration e.g. as drops.
  • the composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a mammal.
  • kits may comprise one or more antigens in liquid form and one or more lyophilised antigens.
  • the flagellin for use in the invention present in the composition may be fused or conjugated to one or more antigens.
  • kits may comprise two vials, or it may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection.
  • compositions used as therapeutics comprise an immunologically effective amount of polypeptide or encoding nucleic acid, as well as any other components, as needed.
  • 'immunologically effective amount' it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc. ) , the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Where more than one antigen is included in a composition then two antigens may be present at the same dose as each other or at different doses.
  • a composition may include a temperature protective agent, and this component may be particularly useful in adjuvanted compositions (particularly those containing a mineral adjuvant, such as an aluminium salt).
  • a liquid temperature protective agent may be added to an aqueous vaccine composition to lower its freezing point e.g. to reduce the freezing point to below 0°C.
  • the temperature protective agent also permits freezing of the composition while protecting mineral salt adjuvants against agglomeration or sedimentation after freezing and thawing, and may also protect the composition at elevated temperatures e.g. above 40°C.
  • a starting aqueous vaccine and the liquid temperature protective agent may be mixed such that the liquid temperature protective agent forms from 1-80% by volume of the final mixture.
  • Suitable temperature protective agents should be safe for human administration, readily miscible/soluble in water, and should not damage other components (e.g. antigen and adjuvant) in the composition.
  • examples include glycerin, propylene glycol, and/or polyethylene glycol (PEG).
  • PEGs may have an average molecular weight ranging from 200-20,000 Da.
  • the polyethylene glycol can have an average molecular weight of about 300 Da ('PEG-300').
  • the invention provides a composition comprising: (i) one or more flagellin polypeptides(s); and (ii) a temperature protective agent.
  • This composition may be formed by mixing (i) an aqueous composition comprising one or more antigen(s), with (ii) a temperature protective agent.
  • the mixture may then be stored e.g. below 0°C, from 0-20°C, from 20-35°C, from 35-55°C, or higher. It may be stored in liquid or frozen form.
  • the mixture may be lyophilised.
  • the composition may alternatively be formed by mixing (i) a dried composition comprising one or more antigen(s), with (ii) a liquid composition comprising the temperature protective agent.
  • component (ii) can be used to reconstitute component (i).
  • composition may be administered orally and may be in the form of a tablet, capsule or powder.
  • Other ingredients such as vitamin C, for example, may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonisation and survival in vivo.
  • a composition for use in the invention includes a therapeutically effective amount of a flagellin polypeptide as described herein.
  • a therapeutically effective amount of a flagellin polypeptide is sufficient to exert a beneficial effect upon a patient.
  • compositions for use in the invention may comprise pharmaceutically acceptable excipients or carriers.
  • suitable excipients may be found in the reference [104].
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [105].
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
  • suitable diluents include ethanol, glycerol and water.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the composition comprises more than one flagellin polypeptide from the genus Enterococcus for use in treating or preventing cancer.
  • the composition comprises a flagellin polypeptide with SEQ ID NO: 1 and at least one further flagellin polypeptide from the genus Enterococcus.
  • the composition comprises a flagellin polypeptide with SEQ ID NO:1 and at least one flagellin polypeptide from the species Enterococcus gallinarum.
  • the composition comprises a flagellin polypeptide with SEQ ID NO:1 and at least one flagellin polypeptide selected from a group consisting of SEQ ID NO 2-42.
  • the composition can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or 45 flagellin polypeptides from the genus Enterococcus and in particular from the species Enterococcus gallinarum.
  • the compositions for use in the invention comprise less than 50 flagellin polypeptides from within the same species (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), and, optionally, do not contain flagellin polypeptides from any other species.
  • compositions for use in the invention comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 flagellin polypeptides from within the same species and, optionally, do not contain flagellin polypeptides from any other species.
  • the individual flagellin polypeptides may be for separate, simultaneous or sequential administration.
  • the composition may comprise all of the flagellin polypeptides, or the flagellin polypeptides may be stored separately and be administered separately, simultaneously or sequentially.
  • the more than one flagellin polypeptides are stored separately but are mixed together prior to use.
  • compositions for use in accordance with the invention may or may not require marketing approval.
  • compositions for use in the invention can comprise pharmaceutically acceptable excipients, diluents or carriers.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is breast cancer.
  • the cancer is mammary carcinoma.
  • the cancer is stage IV breast cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is lung cancer.
  • the cancer is lung carcinoma.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is liver cancer.
  • the cancer is hepatoma (hepatocellular carcinoma).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as described herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is colon cancer.
  • the cancer is colorectal adenocarcinoma.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as described herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is carcinoma.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as described herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is a non-immunogenic cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as described herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is a immunogenic cancer.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as described herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of non-small-cell lung carcinoma, small-cell lung carcinoma, squamous-cell carcinoma, adenocarcinoma, glandular tumors, carcinoid tumors undifferentiated carcinomas.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as described herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancer resulting from a viral infection.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a flagellin polypeptide as described herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: flagellin polypeptide as described herein; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the flagellin polypeptide is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoi
  • a dose of a nucleic acid may have ⁇ 100 ⁇ g nucleic acid; e.g. from 10-100 ⁇ g, such as about 10 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g or 100 ⁇ g, but expression can be seen at much lower levels; e.g. using ⁇ 1 ⁇ g/dose, ⁇ 100ng/dose, ⁇ 10ng/dose, ⁇ 1ng/dose, etc.
  • a dose of a protein antigen may have ⁇ 100 ⁇ g protein; e.g. from 10-100 ⁇ g, such as about 10 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g or 100 ⁇ g.
  • the polypeptide may be administered in a dose of about 0.1-10mg/kg body weight.
  • the pharmaceutical compositions of the invention or the compositions for use in the invention may be administered in a single dose or in more preferably in repeated doses. Preferably repeat doses are administered about every 2, 5, 10, 7, 10 or 15 days.
  • the invention provides the above pharmaceutical composition, wherein the composition is administered at a dose of 100 ⁇ g/kg, 200 ⁇ g/kg, 300 ⁇ g/kg, 400 ⁇ g/kg, 500 ⁇ g/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 50 mg/kg, 100 mg/kg, 200mg/kg or 500mg/kg of subjects body weight.
  • Suitable unit doses include 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, 50 mg, 100 mg, 200mg, 500mg, 1000mg, 100-500 mg, 500-1000mg, 200-400mg, 5-00-750mg, 750-1000mg.
  • the invention provides the above pharmaceutical composition comprising host cells for use in the invention.
  • a suitable daily dose of the host cells may be from about 1 ⁇ 10 3 to about 1 ⁇ 10 11 colony forming units (CFU); for example, from about 1 ⁇ 10 7 to about 1 ⁇ 10 10 CFU; in another example from about 1 ⁇ 10 6 to about 1 ⁇ 10 10 CFU.
  • CFU colony forming units
  • the composition contains the host cell in an amount of from about 1 ⁇ 10 6 to about 1 ⁇ 10 11 CFU/g, respect to the weight of the composition; for example, from about 1 ⁇ 10 8 to about 1 ⁇ 10 10 CFU/g.
  • the dose may be, for example, 1 g, 3g, 5g, and 10g.
  • the composition comprises a mixture of live host cells and host cells that have been killed.
  • the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, sublingual, subcutaneous, intravenous, and intramuscular.
  • the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol.
  • the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water.
  • the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.
  • an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.
  • the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.
  • the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • the invention provides the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4 ° C or about 25 ° C and the container is placed in an atmosphere having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.
  • the composition for use in the invention is provided in a sealed container comprising a composition as described herein.
  • the sealed container is a sachet or bottle.
  • the composition for use in the invention is provided in a syringe comprising a composition as described herein.
  • composition of the present invention may, in some embodiments, be provided as a pharmaceutical formulation.
  • the composition may be provided as a tablet or capsule.
  • the capsule is a gelatine capsule ("gel-cap").
  • compositions for use inthe invention are administered orally.
  • Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth.
  • compositions suitable for oral administration include solid plugs, solid microparticulates, semi-solid and liquid (including multiple phases or dispersed systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), emulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.
  • solid plugs solid microparticulates, semi-solid and liquid (including multiple phases or dispersed systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), emulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.
  • the pharmaceutical formulation is an enteric formulation, i.e. a gastro-resistant formulation (for example, resistant to gastric pH) that is suitable for delivery of the composition for use inthe invention to the intestine by oral administration.
  • enteric formulations may be particularly useful when the bacteria or another component of the composition is acid-sensitive, e.g. prone to degradation under gastric conditions.
  • the enteric formulation comprises an enteric coating.
  • the formulation is an enteric-coated dosage form.
  • the formulation may be an enteric-coated tablet or an enteric-coated capsule, or the like.
  • the enteric coating may be a conventional enteric coating, for example, a conventional coating for a tablet, capsule, or the like for oral delivery.
  • the formulation may comprise a film coating, for example, a thin film layer of an enteric polymer, e.g. an acid-insoluble polymer.
  • the enteric formulation is intrinsically enteric, for example, gastro-resistant without the need for an enteric coating.
  • the formulation is an enteric formulation that does not comprise an enteric coating.
  • the formulation is a capsule made from a thermogelling material.
  • the thermogelling material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC).
  • the capsule comprises a shell that does not contain any film forming polymer.
  • the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [106]).
  • the formulation is an intrinsically enteric capsule (for example, Vcaps ® from Capsugel).
  • the formulation is a soft capsule.
  • Soft capsules are capsules which may, owing to additions of softeners, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, present in the capsule shell, have a certain elasticity and softness.
  • Soft capsules can be produced, for example, on the basis of gelatine or starch. Gelatine-based soft capsules are commercially available from various suppliers.
  • soft capsules can have various shapes, they can be, for example, round, oval, oblong or torpedo-shaped.
  • Soft capsules can be produced by conventional processes, such as, for example, by the Scherer process, the Accogel process or the droplet or blowing process.
  • composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
  • flagellin polypeptides from the genus Enterococcus encompasses wild type flagellin polypeptides and mutated flagellin polypeptides that exhibit immunostimulatory properties, for example which can effectively activate TLR5 responses.
  • flagellin polypeptides also encompasses fragments of wild type flagellin polypeptides and mutated flagellin polypeptides that exhibit immunostimulatory properties, for example which can effectively activate TLR5 responses.
  • references to a percentage sequence identity between two protein sequences means that, when aligned, that percentage of amino acid residues are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in ref. [115].
  • references to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [116].
  • a preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • the Smith-Waterman homology search algorithm is disclosed in ref. [117].
  • sequence identity is calculated over the length of the reference sequence (for example SEQ ID NO:1) and not over the length of the query sequence. Therefore, short and irrelevant sequences are not considered to have high sequence identity to the sequences of the invention.
  • a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps.
  • HEK-Blue TLR5 cells are HEK293 cells co-transfected with the human TLR5 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene.
  • SEAP secreted embryonic alkaline phosphatase
  • the SEAP gene is placed under the control of the IFN- ⁇ minimal promoter fused to five NF- ⁇ B and AP-1-binding sites. Stimulation with a TLR5 ligand activates NF- ⁇ B and AP-1 which induce the production of SEAP.
  • the level of alkaline phosphatase thus relates to the TLR5 activity.
  • Reporter cell line HEK-Blue hTLR5 (Invivogen, hkb-htlr5)
  • Cell growth media DMEM (Sigma, D6171) supplemented with 10% (v/v) FBS (Sigma, F9665), 4mM L-glutamine (Sigma, G7513), 100U/ml penicillin and 100ug/ml streptomiycin (Sigma, P4333), 100ug/ml normocin (Invivogen, ant-nr-1) and selective antibiotics blasticidin (30ug/ml, Invivogen, ant-bl-05) and zeocin (100ug/ml, Invivogen, ant-zn-1)
  • Antibiotic-free media DMEM supplemented with 10% (v/v) FBS and 4mM L-glutamine
  • Bacterial growth conditions All Enterococcus gallinarum MRx0518 cultures were grown in yeast extract-casein hydrolysate-fatty acids (YCFA) broth (E&O Laboratories, UK) at 37°C, under anaerobic conditions.
  • yeast extract-casein hydrolysate-fatty acids (YCFA) broth E&O Laboratories, UK
  • Stationary phase MRx0518 cells 16- 18 hour cultures of strain MRx0518 were grown under the conditions described above using a 1% inoculum of MRx0518 cells, stored frozen as working cell banks at -80°C.
  • Late-log phase MRx0518 cells 3 hour (approx.) cultures of strain MRx0518 were grown under the conditions described above using a 10% inoculum of stationary phase MRx0518 cells.
  • the TLR5 activation assay was performed with the following treatments at two dosages - 10:1 (3 ⁇ 10 5 bacteria) or 100:1(3 ⁇ 10 6 bacteria):
  • a positive control was performed with ultrapure FLA-ST at a concentration of 20ng/ml and 5ng/ml
  • a negative control were performed with antibiotic-free media containing no reporter cells to control for the production of alkaline phosphatase by anything other than the reporter cells.
  • Two additional controls were performed with YCFA and water. For all treatments three replicates were performed.
  • Live and CFS treatments were both prepared from one 10 ml culture of late-log phase MRx0518 cells following centrifugation of the cells at 5000xg for 5mins at room temperature.
  • the bacterial pellet was washed once in PBS (Sigma, D8537) and resuspended in antibiotic-free media to an estimated density of 1.4 ⁇ 10 8 CFU/ml (2-fold dilution) and 1.4 ⁇ 10 7 CFU/ml (20-fold dilution).
  • the bacterial supernatant was collected, filtered (0.22um) under sterile conditions and was diluted 10-fold and 20-fold using H 2 O (Sigma, W3500) to provide equivalents for the bacteria suspensions outlined above.
  • the CFS contains MRx0518 flagella shed into the supernatant.
  • HK cells were generated by incubating a 10 ml culture of late-log phase MRx0518 cells at 80°C for 40 mins. Following centrifugation (conditions described above) the culture supernatant was discarded and the cell pellet was washed once in PBS (Sigma, D8537) and resuspended in antibiotic-free media to an estimated density of 1.4 ⁇ 10 8 CFU/ml (2-fold dilution) and 1.4 ⁇ 10 7 CFU/ml (20-fold dilution). Once prepared, all treatments were stored on ice prior to use. Viable cell counts per ml were determined for all live and heat-killed MRx0518 cell preparations.
  • a known agonist of TLR5 the S. typhimurium flagellin (ultrapure, FLA-ST ultrapure from Invitrogen, tlrl-epsitfla) was prepared at 200 ng/ml or 50 ng/ml in H 2 O.
  • 20 ⁇ l of the appropriate treatment (bacterial cells, cell-free supernatant, positive control ligands, recombinant flagellin, negative controls e.g. YCFA, antibiotic-free media, H 2 O, PBS) were plated in duplicate or triplicate in 96-well cell culture plates (Sigma, CLS3596). 180 ⁇ l of reporter cell suspension was added to each well to give a final volume of 200ul and a final concentration HEK-blue TLR5 cells of 3 ⁇ 10 4 . The assays were incubated at 37°C, 5% CO 2 for 22 h.
  • Quantiblue Invivogen, rep-qb1
  • substrate in a new 96-well plate
  • Quantiblue-containing plates were imaged on a microplate reader (Bio-Rad, iMark) taking 655 nm optical density readings. Results were averaged from technical replicates and then independent experiments averaged thereafter to provide data for final graphs.
  • TLR5 activity was observed after treatment with MRx0518-CFS and was even higher than the known TLR5 agonist FLA-ST ( Figure 6 ).
  • the CFS contains MRx0518 flagella shed into the supernatant.
  • Example 2 - TLR5 activation by the CFS is particularly strong for MRx0518
  • the TLR5 activation assay was performed with the following treatments at two dosages - 10:1 and 100:1:
  • MRx0518-CFS was able to stimulate a high TLR5 response.
  • DSM100110-CFS also produced a TLR5response at an MOI of 100:1, although it was reduced relative to MRx0518-CFS.
  • the MRx0518-CFS produces a higher TLR5 response compared to DSM100110-CFS, potentially because MRx0518 has an improved ability to shed its flagellin into the supernatant compared to other Enterococcus gallinarum strains, which may make it particularly useful in therapy.
  • flagellin polypeptides from the genus Enterococcus may be effective for use in treating or preventing cancer, in particular cancers that are associated with TLR5, such as breast, colorectal and gastric cancer.
  • the TLR5 activation assay was performed with the following treatments at two dosages - 10:1 and 100:1 alone or in combination with trypsin:
  • CFSs were digested with 500 ⁇ g/ml trypsin (0.22um filtered, Sigma, T3924), or an equivalent volume of trypsin vehicle (Hank's balanced salt solution, Fisher Scientific, 1175129) as a mock digest control, for 1hr at 37°C, 5% CO2. After incubation digested and mock digested CFS samples were supplemented with FBS to a final concentration of 10% (v/v) to inhibit trypsin activity.
  • trypsin vehicle Hank's balanced salt solution, Fisher Scientific, 1175129
  • Three negative controls were performed with antibiotic-free media, HBSS and water.
  • a positive control was performed with ultrapure FLA-ST at a concentration of 20ng/ml.
  • TLR5 activation by MRx0518-CFS was abolished by treatment with trypsin ( Figure 8 ).
  • Figure 8 show that the TLR5 response is activated by proteins in the MRx0518-CFS. Degradation of these proteins by trypsin abolishes the TLR5 response.
  • the MRx0518 flagellin protein (FlaA MRx0518 ) contains 36 trypsin cleavage sites, some of which are located in the TLR5 interaction domains.
  • Recombinant flagellin proteins, FlaA MRx0518 and FlaA DSM100110 were expressed in E.coli cells and purified using an Immobilized Metal Affinity Chromatography and imidazole elution, followed by endotoxin removal (see table below). This results in high purity preparations with minimal endotoxin levels. The size and purity of the proteins was confirmed using an SDS-PAGE gel ( Figure 9b ).
  • the TLR5 activation assay was performed with the following treatments:
  • a negative control was performed with a FLA vehicle at a concentration 5ng/ml and a positive control was performed with ultrapure FLA-ST at a concentration of 20ng/ml. For each treatment three replicates were performed.
  • Both recombinant FlaA MRx0518 and FlaA DSM100110 can activate TLR5 responses.
  • Figure 10 compares the TLR5 activation with different dosages of recombinant FlaA MRx0518 and FlaA DSM100110 with the treatments described in examples 3 and 4.
  • both recombinant flagellin proteins can produce a TLR5 response that is higher than produced by the well-known TLR5 agonist FLA-ST being used a higher concentration (20ng/ml).
  • flagellin polypeptides from Enterococcus gallinarum are more effective than flagellin polypeptides from S. typhimurium at treating or preventing cancer.
  • FlaA MRx0518 and FlaA DSM100110 have different immunostimulatory profiles. At concentrations of 1ng/ml and 0.2ng/ml FlaA MRx0518 produces approximately five times more potent TLR5 response than FlaA DSM100110 . Both MRx0518 and DSM100110 are highly related strains. Both are flagellated Enterococcus gallinarum strains, however they express fundamentally different flagellin that have different immunostimulatory profiles. The surprising finding that FlaA MRx0518 is strong TLR5 activator shows that this flagellin polypeptide is particularly effective at treating or preventing cancer.
  • flagellin polypeptides from the genus Enterococcus are agonists for TLR5.
  • TLR5 agonists have been implicated in treating T and B cell lymphomas. Therefore, flagellin polypeptides from the genus Enterococcus may be particularly effective at treating or preventing T and B cell lymphomas.
  • TLR5 activation has been shown to ameliorate radiation induced tissue damage.
  • the examples show that flagellin polypeptides from the genus Enterococcus are strong activators of the TLR5 response. Therefore, flagellin polypeptides from the genus Enterococcus, and in particular the species Enterococcus gallinarum, may be particularly effective at ameliorating radiation induced tissue damage.
  • NlRx0518 An insertion mutant of NlRx0518 was created which disrupted the flagellin gene.
  • the TLR5 activation assay was performed with the following treatments at two dosages - 10:1 and 100:1:
  • Positive controls were performed with ultrapure FLA-ST at a concentrations of 5ng/ml and 20ng/ml.
  • a negative control was performed with YFCA at two dosages - 10:1 and 100:1. For all treatments three replicates were performed.
  • Bacteria morphology such as size, presence or absence of fimbrae/ flagella/pilli, or the presence of extracellular matrix influence both motility and adherence and are therefore important in the characterization of bacteria.
  • MRx0518 cultures were examined at the electron microscope level (Scanning and Transmission) to allow visualization of bacteria structure at a higher resolution and magnification than that allowed by conventional light microscopy methods.
  • Example 8 Activation of murine TLR5 by recombinant flagellin proteins from Enterococcus gallinarum strains MRx0518 and DSM 100110
  • MRx0518 is a bacterial strain isolated from a human sample, whereas DSM 100110 is of murine origin. The ability of the recombinant flagellin proteins from the Enterococcus gallinarum strains MRx0518 and DSM 100110 to activate murine TLR5 was tested.
  • Recombinant purified MRx0518 (FliC MRx0518 ) and DSM 100110 (FliC DSM100110 ) were produced as described in Example 4.
  • TLR5 assay as described in Example 1 was performed.
  • the TLR5 activation assay was performed with the following treatments:
  • the different activation profiles of human and murine TLR5 by recombinant flagellin proteins from different Enterococcus gallinarum strains may be due to sequence differences between FliC MRx0518 and FliC DSM100110 , as well as binding site variability between human and murine TLR5.
  • the MRX and test strains were isolated from one of four donors from the 4D Pharma culture collection (donor numbers F00, F14, F19 and F22).
  • the bacterial strains were cultured in YCFA broth (E&O Laboratories, Bonnybridge, Scotland, UK) until they reached stationary growth phase. Cells and supernatants were separated by centrifugation at 5,000 x g for 5 mins. Supernatants were passed through a 0.22 ⁇ m filter and diluted in water.
  • HEK-Blue TM -hTLR5 cells (InvivoGen, San Diego, CA, USA) were routinely cultured in DMEM supplemented with 10 % FBS, 4 mM L-glutamine, 4.5 mg/ml glucose, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 100 ⁇ g/ml Normocin TM (InvivoGen), 30 ⁇ g/ml blastocidin (InvivoGen) and 100 ⁇ g/ml zeocin (InvivoGen) to 90 % density.
  • Cell lines were cultured at 37 °C and 5 % CO 2 . All reagents were supplied by Sigma-Aldrich, Gillingham, England, UK unless otherwise stated.
  • MRx0518 The ability of the bacterial strain MRx0518 to activate NF- ⁇ B was investigated. The results are presented in Figure 18 . MRx0518 supernatant was the most potent activator of NF- ⁇ B. The activation of NF- ⁇ B was eliminated after treatment with trypsin.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs were plated in 96-well plates plated with anti-CD3 (Ebioscience, CD3 monoclonal antibody (OKT3 clone), functional grade, cat. No. 16-0037-81) at 400,000/well in 50 ⁇ l cRPMI medium per well (cRPMI contains RPMI 1640 (+L-Glut, 21875-034) 2mM final conc. Stock 200mM.; 10% HIFBS (Gibco life technologies, 10082-147); 50 ⁇ m mercaptoethanol (Gibco life technologies, 21985-023); and 1% pen/strep (P4333, 10mg/ml). MRx518 supernatant was then added to each well, 4,000,000 in 100 ⁇ l/well. Supernatants were passed through a 0.22 ⁇ m filter and diluted appropriately in co-culture.
  • the cells were removed and re-suspended in a medium containing PMA- (Sigma, Cat no. P8139), Ionomycin (Sigma, Cat no. 13909) and GolgiSTOP (BD, Cat no 554724) for 5 hours.
  • PMA stock was 1mg/ml in DMSO which was further diluted in 100ug/ml (each sample required 50ng/ml in cRPMI), Ionomycin stock was 1mM in DMSO (1 ⁇ M in cRPMI was used) and GolgiStop concentration was used at 4 ⁇ 1/6ml.
  • the cells were then subjected to a flow cytometry staining: After washing, the cells were incubated with Viobility 405/520 Fixable Dye from Miltenyi biotec (1 ⁇ l/sample) + human Fc block, cat. 564219 (1 ⁇ l/sample) in PBS for 10 mins in the dark at room temperature. The surface antibodies (2 ⁇ l of each) were then added directly to the wells for 10 mins in the dark at room temperature - CD3-APC-Vio 770 (Miltenyi, cat. No. 130-113-136), CD4-VioBlue (Miltenyi, cat. No. 130-114-534) and CD25-VioBright FITC (Miltenyi, cat. No. 130-113-283). The cells were then washed twice in PBS and spun down at 300g/5min/RT.
  • the eBioscience FoxP3 transcription factor staining buffer was then used to fix and permeabilise the cells (cat. No. 00-5523). Following the eBioscience protocol, a perm/fix buffer was prepared using 1x concentrate and 3 diluent. The cells were fixed for 1h at RT and then washed 2x in 1x Perm wash and spun down at 300g/5min/RT.
  • Intracellular markers Transcription factors 2ul IL10-PE 5.5ul FoxP3-PE-Cy7 2ul IFNy-PE Vio770 9ul Tbet-APC 10ul IL,17a-APC 9ul RoRyt-PE
  • MRx518 supernatant can induce the differentiation of T helper cells and cytotoxic T cells.
  • This example provides an alternative strategy for inactivating the flagellin gene compared to Example 5.
  • the ⁇ liC gene was disrupted by homology-driven insertion of the suicide plasmid pORI19 (see Figure 20 ). Insertion of pORI19 within the ⁇ liC gene was confirmed by DNA sequencing and the non-motile phenotype of the resulting mutant strain was confirmed in vitro.
  • the flagellin insertion mutant was created using the non-replicative plasmid pORI19 (Emr repA- Ori+; cloning vector [118]).
  • An internal fragment of E . gallinarum MRx0518 fliC gene was amplified using primers DC020 (SEQ ID NO: 43: CCCGGGGGATCCGCGGTAAATGTTGCTAAAGCATCATCG) and DC021 (SEQ ID NO: 44: ACGACGGTCGACCCACAGCATCTTAGGGCGTATGCG) and cloned into pORI19. Restriction enzymes and Quick Ligase (New England Biolabs, Ipswich, MA, USA) were used according to the manufacturer's instructions. This construct was propagated in E.
  • coli EC101 by chemical transformation [118] and isolated using the Genopure Plasmid Maxi Kit (Roche Diagnostics, Basel, Switzerland) from a 500-ml culture. Isolated plasmid DNA was concentrated using 0.3 M sodium acetate pH 5.2 and ethanol down to 20 ⁇ l.
  • Electrocompetent cells were successfully generated by growing bacterial cultures in sub-inhibitory concentrations of glycine followed by mutanolysin and lysozyme treatments to further weaken the cell wall peptidoglycan layer.
  • a protocol was developed to prepare E. gallinarum MRx0518 electrocompetent cells, which was adapted from a previously published method [119].
  • E . gallinarum MRx0518 was grown for 18 h in GM17 broth, supplemented with 0.5 M sucrose and 3 % (w/v) glycine (Sigma-Aldrich).
  • E . gallinarum MRx0518 cells were then transformed by electroporation with 10 ⁇ g of plasmid DNA and recovered in BHI broth before plating on selective BHI agar. Positive colonies were screened for the presence of the em gene using primers DC047 (SEQ ID NO:45: CCAAATTAAAGAGGGTTATAATGAACGAG) and DC048 (SEQ ID NO:46: GATGCAGTTTATGCATCCCTTAAC).
  • Plasmid insertion was confirmed for successful transformants by PCR amplification and sequencing (GATC Biotech, Konstanz, Germany) using primers DC013 (SEQ ID NO:47: CCGATAAATAGTAGCAGAGGGAAACC) and DC014 (SEQ ID NO:48: GGCTGAATATCCATCAGAGCTTCCTC).

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RS20220288A RS63090B1 (sr) 2018-03-19 2019-03-19 Kompozicije entrecoccus flagelina za upotrebu u terapiji
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SI201930190T SI3768284T1 (sl) 2018-03-19 2019-03-19 Sestavki iz flagelina enterococcusa za uporabo v terapiji
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US20230022045A1 (en) * 2019-12-03 2023-01-26 Board Of Regents, The University Of Texas System Combination therapies for the treatment of cancer
WO2021252632A2 (en) * 2020-06-09 2021-12-16 Flagship Pioneering, Inc. Physiologically acceptable compositions containing microorganisms or microbial products
EP4384194A1 (en) * 2021-10-08 2024-06-19 Iowa State University Research Foundation, Inc. Use of dopamine producing products to increase vaccine efficacy
CN113999923B (zh) * 2021-11-25 2024-05-14 上海金翌生物科技有限公司 小柳-原田综合症标志微生物及其应用
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR106769A1 (es) * 2015-11-20 2018-02-14 4D Pharma Res Ltd Composiciones que comprenden cepas bacterianas

Family Cites Families (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US4500707A (en) 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US5132418A (en) 1980-02-29 1992-07-21 University Patents, Inc. Process for preparing polynucleotides
US4973679A (en) 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
US4668777A (en) 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4373071A (en) 1981-04-30 1983-02-08 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US5153319A (en) 1986-03-31 1992-10-06 University Patents, Inc. Process for preparing polynucleotides
US5262530A (en) 1988-12-21 1993-11-16 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5047524A (en) 1988-12-21 1991-09-10 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
JPH08259450A (ja) 1995-03-17 1996-10-08 Nichinichi Seiyaku Kk インターフェロン産生増強剤
US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
EP0904784A1 (en) 1997-09-22 1999-03-31 N.V. Nutricia Probiotic nutritional preparation
GB0127916D0 (en) 2001-11-21 2002-01-16 Rowett Res Inst Method
US20040009937A1 (en) * 2002-01-31 2004-01-15 Wei Chen Methods and composition for delivering nucleic acids and/or proteins to the respiratory system
US20080025977A1 (en) 2003-04-14 2008-01-31 Arius Research, Inc. Cytotoxicity mediation of cells evidencing surface expression of CD59
US20060228369A1 (en) 2005-04-11 2006-10-12 Program For Appropriate Technology In Health Stabilization and preservation of temperature-sensitive vaccines
JP2007116991A (ja) 2005-10-28 2007-05-17 Eternal Light General Institute Inc 機能性食品
ES2895666T3 (es) 2006-10-27 2022-02-22 Capsugel Belgium Nv Cápsulas duras de hidroxipropilmetilcelulosa y proceso de fabricación
EP1958647A1 (en) 2007-02-15 2008-08-20 Helmholtz-Zentrum für Infektionsforschung GmbH Pharmaceutical composition with bacteria for tumor treatment
EP2133092B1 (en) 2007-03-19 2015-12-09 Morishita Jintan Co., Ltd. Oral vaccine
JP2011519834A (ja) * 2008-04-25 2011-07-14 インスティチュート フォー システムズ バイオロジー フラジェリンポリペプチドワクチン
KR20120104177A (ko) * 2009-10-06 2012-09-20 파나셀라 랩스, 아이엔씨. 암을 치료하기 위한 톨 유사 수용체 및 효능제의 용도
EP2663367A4 (en) 2011-01-10 2014-08-06 Cleveland Biolabs Inc USE OF A TOLL-TYPE RECEPTOR AGONIST FOR THE TREATMENT OF CANCER
ES2877100T3 (es) 2011-07-13 2021-11-16 Pharmaxis Ltd Mejoras relacionadas con los dispositivos de suministro
GB201112091D0 (en) 2011-07-14 2011-08-31 Gt Biolog Ltd Bacterial strains isolated from pigs
GB201117313D0 (en) 2011-10-07 2011-11-16 Gt Biolog Ltd Bacterium for use in medicine
ES2408279B1 (es) 2011-12-15 2014-09-09 Universidad De Las Palmas De Gran Canaria Bacteria acido láctica probiótica
FR2989002B1 (fr) 2012-04-10 2015-05-15 Beepratte Lab Compositions a base de probiotiques et d'un complexe beepollen/argile, leur preparation et leurs utilisations en nutrition et therapeutique
WO2014043593A2 (en) 2012-09-13 2014-03-20 Massachusetts Institute Of Technology Programmable drug delivery profiles of tumor-targeted bacteria
GB201306536D0 (en) 2013-04-10 2013-05-22 Gt Biolog Ltd Polypeptide and immune modulation
EP3104885A4 (en) * 2014-02-12 2017-08-30 Transalgae (Israel) Ltd Algal based edible vaccines
GB201520497D0 (en) 2015-11-20 2016-01-06 4D Pharma Res Ltd Compositions comprising bacterial strains
EP3209310B1 (en) 2015-11-20 2018-01-31 4D Pharma Research Limited Compositions comprising bacterial strains
TW201907931A (zh) * 2017-05-24 2019-03-01 英商4D製藥研究有限公司 包含細菌菌株之組合物
BR112019028301A2 (pt) 2017-07-05 2020-07-14 Evelo Biosciences, Inc. composições e métodos para tratar câncer com o uso de bifidobacterium animalis ssp. lactis
TW201919674A (zh) 2017-08-10 2019-06-01 英商4D製藥研究有限公司 包含細菌菌株之組合物
JP2021518129A (ja) 2018-03-19 2021-08-02 フォーディー ファーマ リサーチ リミテッド４Ｄ Ｐｈａｒｍａ Ｒｅｓｅａｒｃｈ Ｌｉｍｉｔｅｄ 組成物

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR106769A1 (es) * 2015-11-20 2018-02-14 4D Pharma Res Ltd Composiciones que comprenden cepas bacterianas

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BARTHOLOMEUS VAN DEN BOGERT ET AL: "Immunomodulatory Properties of Streptococcus and Veillonella Isolates from the Human Small Intestine Microbiota", PLOS ONE, vol. 9, no. 12, 5 December 2014 (2014-12-05), pages e114277, XP055525331, DOI: 10.1371/journal.pone.0114277 *

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